Factor IX of the blood coagulation system: a review

Factor IX is a factor of the blood coagulation system. Its activation occurs on the surface of phospholipid membranes. It can be activated by the factor VIIa-TF (tissue factor)-Ca2+ complex via an extrinsic pathway and by factor XIa in the presence of Ca2+ via the intrinsic pathway of blood coagulation system activation. The activated factor IXa is a serine proteinase. The main function of the activated factor IXa in complex with factor VIIIa and phospholipids in presence of Ca2+ consists of the activation of factor X. Factor IX is synthesized in the liver and is subject to a number of posttranslational modifications including gamma-carboxylation, beta-hydroxylation, and glycosylation. It forms a subgroup of vitamin K-dependent plasma proteins including factors VII and X and protein C characterized by identical domain structures having high levels of homology. Factor IX consists of an NH2-terminal Gla domain, two epidermal growth factor (EGF)-like domains, and a C-terminal domain containing Ser in its active site. Factor IX deficiency in human plasma results in the disease known as hemophilia B.     

Thrombin activates factor XI on activated platelets in the absence of factor XII

Thrombin can activate factor XI in the presence of dextran sulfate or sulfatides. However, a physiological cofactor for thrombin activation of factor XI has not been identified. We examined this question in a cell-based, tissue factor-initiated model system. In the absence of factor XII, factor XI enhanced thrombin generation in this model. The effect on thrombin generation was reproduced by 2 to 5 pmol/L factor XIa. A specific inhibitor of factor XIIa did not diminish the effect of factor XI. Thus, factor XI can be activated in a model system that does not contain factor XIIa or nonphysiological cofactors. Preincubation of factor XI with activated platelets and thrombin or factor Xa enhanced subsequent thrombin generation in the model system. Preincubation of factor XI with thrombin or factor Xa, but without platelets, did not enhance thrombin generation, suggesting that these proteases might activate factor XI on platelet surfaces. Thrombin and factor Xa were then directly tested for their ability to activate factor XI. In the presence of dextran sulfate, thrombin or factor Xa activated factor XI. Thrombin, but not factor Xa, also cleaved detectable amounts of factor XI in the presence of activated platelets. Thus, thrombin activates enough factor XI to enhance subsequent thrombin generation in a model system. Platelet surfaces might provide the site for thrombin activation of functionally significant amounts of factor XI in vivo.     

Antioxidant properties of the decarboxylated dimer of aminoethylcysteine ketimine: assessment of its ability to scavenge peroxynitrite

In the present study we used an in-vitro technique to examine initiation and propagation of blood coagulation at the surface of tantalum coronary stents exposed to flowing platelet-rich and platelet-free plasma. The time course of factor IXa production at the surface of the stent was not influenced by platelets. In spite of a significant factor IXa production, no thrombin activity was detected when the tantalum stent was exposed to platelet-free plasma; only when the stent was exposed to platelet-rich plasma was extensive thrombin production observed. These findings indicate that tantalum triggers blood coagulation, but that (adherent) platelets are essential for thrombin generation. Heparin-coated tantalum stents exposed to flowing platelet-rich plasma showed that factor IXa generation was slightly reduced compared with the bare stent. However, the heparin coating drastically delayed the onset of thrombin generation and largely reduced the steady-state production of thrombin. We found a clear relationship between the antithrombin binding capacity and the antithrombogenic potential of the heparin-coated stents. The mode of action of immobilized heparin is thought to abrogate thrombin generation by inhibiting thrombin-dependent positive feedback reactions at the surface of the coronary stent.     

Neutrophil elastase cleavage of human factor IX generates an activated factor IX-like product devoid of coagulant function

In preliminary studies, the generation of thrombin in vivo was found to induce a 92% loss of functional activity of factor IX (F.IX) despite the detection by Western blotting of a product resembling activated F.IX (F.IXa) and a 25% increase in F.IX antigen levels (Hoogendoorn et al, Thromb Haemost 69:1127, 1993 [abstr]). These changes were associated with evidence of increased elastase availability. To study the possibility that these two observations were related, a detailed physical and functional characterization of the hydrolysis of purified human F.IX by human neutrophil elastase (HNE) was performed in vitro. An activated partial thromboplastin time (aPTT) clotting assay demonstrated that, although HNE eliminated the potential of F.IX to be activated, it only marginally reduced the F.IXa activity. Reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that HNE treatment of F.IX generated cleavage products of 30 and 20 kD that could not be distinguished from the respective heavy and light chain peptides that were identified in parallel studies when F.IX was activated by activated bovine F.XI (F.XIa), one of its physiological activators. In addition, nonreducing SDS-PAGE demonstrated that HNE-treated F.IX formed no complexes with antithrombin III (ATIII) in the presence of heparin. Furthermore, HNE-treated F.IX was unable to (1) bind the active site probe p-aminobenzamidine; (2) hydrolyze the synthetic peptide substrate CH3SO2-Leu-Gly-Arg-p-nitroanilide; and (3) activate human factor X (F.X). In contrast to dansyl-Glu-Gly-Arg-chloromethyl ketone (dEGR)-inactivated F.IXa, HNE-treated F.IX (0.01 to 10,000 pmol/L) failed to inhibit the clotting activity of F.IXa (10 pmol/L) in the aPTT. NH2-terminal sequencing indicated that HNE cleaved human F.IX at Thr140, Thr144, Ile164, Thr172, and Val181. The cleavages at Thr140/Thr144 and at Thr172/Val181 are both very close to the normal F.XIa alpha-(Arg145) and beta-(Arg180) cleavage sites, respectively. In summary, the results suggest that the activatability of F.IX is eliminated after cleavage by HNE and that the inability of HNE-treated F.IX to support F.IXa-like coagulant function is a consequence of improper active site formation. These in vitro observations support the possibility that increased HNE cleavage of F.IX in vivo may contribute to the disregulation of hemostasis that occurs in conditions such as disseminated intravascular coagulation (DIC).     

Polyserositis in a female patient with hypothyroidism

Advances in our knowledge of the biochemistry of coagulation have facilitated the development of sensitive and specific assays that are able to detect the generation of coagulation enzymes in vivo. It has been demonstrated that the factor VII-tissue factor pathway functions under normal conditions to generate factor Xa and convert prothrombin to thrombin. Furthermore, the factor VII-tissue factor pathway is also mainly responsible for the activation of factor IX with minimal contribution from the contact phase. However the relatively high levels of factor IXa generated are unable to convert factor X to factor Xa under basal conditions. Prospective studies are required to determine whether "biochemically" hypercoagulable individuals (i.e., those with elevated levels of free factor VIIa, activation peptides of factor IX, factor X, or prothrombin) are more likely to develop arterial or venous thrombosis.     

Identification of a factor IX binding site on the third apple domain of activated factor XI

Activated factor XI (factor XIa) participates in blood coagulation by activating factor IX. Previous work has demonstrated that a binding site for factor IX is present on the noncatalytic heavy chain of factor XIa (Sinha, D., Seaman, F. S., and Walsh, P. N. (1987) Biochemistry 26, 3768-3775). Recombinant factor XI proteins were expressed in which each of the four apple domains of the heavy chain (designated A1 through A4) were individually replaced with the corresponding domain from the homologous but functionally distinct protease prekallikrein (PK). To identify the site of factor IX binding, the chimeric proteins were activated with factor XIIa and tested for their capacity to activate factor IX in plasma coagulation and purified protein assays. The chimera with the substitution in the third apple domain (factor XI/PKA3) had <1% of the coagulant activity of wild type factor XIa in a plasma coagulation assay, whereas the chimeras with substitutions in A1, A2, and A4 demonstrated significant activity (68-140% of wild type activity). The Km for activation of factor IX by factor XIa/PKA3 (12. 7 microM) is more than 30-fold higher than the Km for activation by wild type factor XIa or the other factor XI/PK chimeras (0.11-0.37 microM). Two monoclonal antibodies (2A12 and 11AE) that recognize epitopes on the factor XI A3 domain were potent inhibitors of factor IX activation by factor XIa, whereas antibodies against the A2 (1A6) and A4 (3G4) domains were poor inhibitors. The data indicate that a binding site for factor IX is present on the third apple domain of factor XIa.     

Blood coagulation in hemophilia A and hemophilia C

Tissue factor (TF)-induced coagulation was compared in contact pathway suppressed human blood from normal, factor VIII-deficient, and factor XI-deficient donors. The progress of the reaction was analyzed in quenched samples by immunoassay and immunoblotting for fibrinopeptide A (FPA), thrombin-antithrombin (TAT), factor V activation, and osteonectin. In hemophilia A blood (factor VIII:C <1%) treated with 25 pmol/L TF, clotting was significantly delayed versus normal, whereas replacement with recombinant factor VIII (1 U/mL) restored the clot time near normal values. Fibrinopeptide A release was slower over the course of the experiment than in normal blood or hemophilic blood with factor VIII replaced, but significant release was observed by the end of the experiment. Factor V activation was significantly impaired, with both the heavy and light chains presenting more slowly than in the normal or replacement cases. Differences in platelet activation (osteonectin release) between normal and factor VIII-deficient blood were small, with the midpoint of the profiles observed within 1 minute of each other. Thrombin generation during the propagation phase (subsequent to clotting) was greatly impaired in factor VIII deficiency, being depressed to less than 1/29 (<1.9 nmol TAT/L/min) the rate in normal blood (55 nmol TAT/L/min). Replacement with recombinant factor VIII normalized the rate of TAT generation. Thus, coagulation in hemophilia A blood at 25 pmol/L TF is impaired, with significantly slower thrombin generation than normal during the propagation phase; this reduced thrombin appears to affect FPA production and factor V activation more profoundly than platelet activation. At the same level of TF in factor XI-deficient blood (XI:C <2%), only minor differences in clotting or product formation (FPA, osteonectin, and factor Va) were observed. Using reduced levels of initiator (5 pmol/L TF), the reaction was more strongly influenced by factor XI deficiency. Clot formation was delayed from 11.1 to 15.7 minutes, which shortened to 9.7 minutes with factor XI replacement. The maximum thrombin generation rate observed ( approximately 37 nmol TAT/L/min) was approximately one third that for normal (110 nmol/L TAT/min) or with factor XI replacement (119 nmol TAT/L/min). FPA release, factor V activation, and release of platelet osteonectin were slower in factor XI-deficient blood than in normal blood. The data demonstrate that factor XI deficiency results in significantly delayed clot formation only at sufficiently low TF concentrations. However, even at these low TF concentrations, significant thrombin is generated in the propagation phase after formation of the initial clot in hemophilia C blood.     

Advances and dilemmas in factor XI

Factor XI is a key component of the intrinsic pathway of blood coagulation in vitro. The poor correlation between the clinical bleeding diathesis in factor XI deficiency and abnormalities in clotting assays that measure intrinsic coagulation brings into question the role of this serine protease in in vivo hemostasis. The characterizations of the point mutations responsible for the majority of cases of severe factor XI deficiency in Ashkenazi Jews and subsequent epidemiologic studies have provided insight into the perplexing hemostatic abnormalities in this disorder. It appears that excessive bleeding in factor XI deficiency depends on the severity of the deficiency in certain situations and on the location of the hemostatic challenge in others. Additional coexisting abnormalities of hemostasis, such as von Willebrand's disease, may also be responsible for variation in clinical presentation, particularly in those individuals with mild factor XI deficiency. The absence of abnormal bleeding in congenital deficiency of factor XII, the protease that activates factor XI in the intrinsic cascade, has stimulated a search for other mechanisms for factor XI activation. Recent studies have pointed to the serine protease thrombin and autoactivation by activated factor XI as possible alternatives to factor XII as activators of factor XI. These findings suggest that factor XI, rather than operating in a pathway for the initiation of hemostasis, may function in the consolidation of clot formation after the initiation of the hemostatic process by other mechanisms.     

Lipoproteins and the haemostatic system in atherothrombotic disorders

The early belief that the haemostatic system has no active role in the formation of the atheromatous plaque is no longer tenable. Rather, the association between hypercholesterolaemia and atherosclerosis appears to arise in part because of various effects of high concentrations of LDL and VLDL particles on the cellular and humoral components of the system, thereby promoting plaque growth and thrombosis. These may be summarized as follows: 1. High concentrations of native LDL have been reported to promote the adhesion of monocytes to the endothelial cell, suggesting that the latter undergoes a form of activation upon such exposure. Oxidized LDL is more potent in this respect, and persistent exposure of endothelium to such particles can eventually lead to cell injury. 2. Activated endothelial cells acquire characteristics on their luminal surface conducive to thrombin generation and fibrin production. Thrombin has several actions on the endothelial cell, monocyte, smooth muscle cell and platelet which in the presence of hypercholesterolaemia will promote the formation of atheroma. 3. Oxidatively modified LDL can activate circulating monocytes, when they also acquire procoagulant properties which favour thrombin production. 4. Platelets show an increased tendency to aggregate when exposed to hypercholesterolaemic plasma. This effect may arise in part because the platelet of the hypercholesterolaemic patient expresses an increased number of fibrinogen binding sites on its surface following activation by agonists such as ADP. These hyperaggregable platelets adhere to activated endothelial cells which express von Willebrand factor on their surface, and to subendothelial proteins exposed in the gaps that open between injured endothelial cells. Platelets exposed to raised LDL levels also show a reduced sensitivity to prostacyclin, an antiaggregatory agent. Oxidatively modified LDL has been reported to stimulate aggregation of platelets in the absence of other agonists such as ADP or thrombin (spontaneous aggregation). 5. Platelet aggregation and fibrin deposition at sites of endothelial injury will create microthrombi which become incorporated into the lesion by organization, thereby increasing the fibrous and cellular content of the atheromatous plaque. 6. Lipolysis of triglyceride-rich lipoproteins at the endothelial cell surface leads to transient activation of the coagulation mechanism with activation of factor VII. Activated factor VII is a potent procoagulant when it forms a complex with tissue factor in the atheromatous lesion. Persistent hypertriglyceridaemia is accompanied by raised concentrations of factor X, factor IX, factor VII and prothrombin. 7. Hypertriglyceridaemia is associated with an increased plasma concentration of PAI-1 and a reduction in plasma fibrinolytic activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Factor XI activation by meizothrombin: stimulation by phospholipid vesicles containing both phosphatidylserine and phosphatidylethanolamine

The activation of factor XI by meizothrombin was investigated using recombinant meizothrombin (R155A meizothrombin) that is resistant to autocatalytic removal of fragment 1. Meizothrombin was capable of activating factor XI at an activation rate similar to that of thrombin. Dextran sulphate and heparin, known cofactors of thrombin-mediated factor XI activation, did not stimulate the activation of factor XI by meizothrombin. However, the activation of factor XI by meizothrombin was markedly enhanced by vesicles containing phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylethanolamine (PE), whereas PC/PS or PC/PE vesicles only had a minor effect on the activation. Thrombin-mediated factor XI activation was not influenced by phospholipids. The effect of PC/PS/PE and PC/PS vesicles was studied in a factor XI dependent clot lysis assay. In this assay, factor XI inhibits clot lysis by a feedback loop in the intrinsic pathway via thrombin-mediated factor XI activation. Removal of endogenous phospholipids in plasma by centrifugation resulted in an increased clot lysis, which could be restored to the pre-centrifugation level by the addition of PC/PS/PE vesicles, but not by PC/PS vesicles. When clot lysis was initiated by factor IXa in the presence of a factor XIa blocking antibody, there was no difference in inhibitory effect of PC/PS/PE or PC/PS vesicles. These data suggested that the differences in clot lysis inhibition observed between PC/PS/PE and PC/PS vesicles were caused by factor XI activation by meizothrombin. Meizothrombin-mediated factor XI activation may therefore play an important role in the antifibrinolytic feedback loop in the intrinsic pathway.     

Platelet functions and haemostasis parameters in pigs: absence of side effects of a procedure of general anaesthesia

Pigs are largely used as experimental animal models of thrombosis and for testing the anti thrombotic drug efficacy. Generally experiments are performed on pigs under general anaesthesia and observations can be affected by the anaesthetic drugs used. The effects of a general anaesthetic procedure were checked on pig haemostasis parameters; the pig was pre-anaesthetized with ketamine chloride, then intubated and ventilated with a mixture containing halothane, nitrous oxide and oxygen. Bleeding time, platelet aggregations, coagulation factors, coagulation inhibitors, fibrinolysis parameters and markers of activation of coagulation were determined on 30 Large White pigs before and under this anaesthesia procedure. Compared to human coagulation, pig is characterized by very high levels of factor V, VIII, IX, XI, XII activities, same levels of factor II, fibrinogen, antithrombin III (ATIII), low levels of protein C activities. Thrombin-antithrombin complex (TAT) and tissue plasminogen activator antigen (tPA) values were dispersed. With the reagents used, protein S, prothrombin fragment 1 + 2 (F1 + 2), D Dimers (D-D), plasminogen activator inhibitor (PAi) levels could not be determined. No difference was observed between results obtained before and under anaesthesia, particularly to increase of bleeding time, no modification of platelet aggregations and no activation of coagulation. This anaesthetic procedure does not induce any modification of pig haemostasis and can be used, without side effects, for experimental thrombosis studies in pigs.     

An in vitro analysis of the combination of hemophilia A and factor V(LEIDEN)

The classification of factor VIII deficiency, generally used based on plasma levels of factor VIII, consists of severe (<1% normal factor VIII activity), moderate (1% to 4% factor VIII activity), or mild (5% to 25% factor VIII activity). A recent communication described four individuals bearing identical factor VIII mutations. This resulted in a severe bleeding disorder in two patients who carried a normal factor V gene, whereas the two patients who did not display severe hemophilia were heterozygous for the factor V(LEIDEN) mutation, which leads to the substitution of Arg506 --> Gln mutation in the factor V molecule. Based on the factor VIII level measured using factor VIII-deficient plasma, these two patients were classified as mild/moderate hemophiliacs. We studied the condition of moderate to severe hemophilia A combined with the factor V(LEIDEN) mutation in vitro in a reconstituted model of the tissue factor pathway to thrombin. In the model, thrombin generation was initiated by relipidated tissue factor and factor VIIa in the presence of the coagulation factors X, IX, II, V, and VIII and the inhibitors tissue factor pathway inhibitor, antithrombin-III, and protein C. At 5 pmol/L initiating factor VIIa x tissue factor, a 10-fold higher peak level of thrombin formation (350 nmol/L), was observed in the system in the presence of plasma levels of factor VIII compared with reactions without factor VIII. Significant increase in thrombin formation was observed at factor VIII concentrations less than 42 pmol/L (approximately 6% of the normal factor VIII plasma concentration). In reactions without factor VIII, in which thrombin generation was downregulated by the addition of protein C and thrombomodulin, an increase of thrombin formation was observed with the factor V(LEIDEN) mutation. The level of increase in thrombin generation in the hemophilia A situation was found to be dependent on the factor V(LEIDEN) concentration. When the factor V(LEIDEN) concentration was varied from 50% to 150% of the normal plasma concentration, the increase in thrombin generation ranged from threefold to sevenfold. The data suggested that the analysis of the factor V genotype should be accompanied by a quantitative analysis of the plasma factor V(LEIDEN) level to understand the effect of factor V(LEIDEN) in hemophilia A patients. The presented data support the hypothesis that the factor V(LEIDEN) mutation can increase thrombin formation in severe hemophilia A.     

Actions of interleukin-4 on prostaglandin biosynthesis by human amnion cells

Acute spinal cord injury (SCI) is associated with a marked propensity to thromboembolism and a variety of coagulation abnormalities. However, data on blood coagulation profiles in patients with uncomplicated long-standing SCI are limited. These data were studied here. Eight men with uncomplicated chronic SCI and nine able-bodied normal men were studied. Plasma activities and/or antigen concentrations of high molecular weight kininogen (HMWK) and of factors XII, XI, IX, VIII, VII, X, V, II and XIII as well as von Willebrand factor (vWF), fibrinogen and fibronectin were measured by appropriate functional and or immunological assays. The SCI group exhibited normal values for factors XII, IX, VIII, vWF, VII, X and V as well as HMWK, vWF and fibronectin concentration. However, they showed slight reductions in plasma factor XI activity, factor XIII antigen concentration and modest increases in fibrinogen and factor II concentrations. No correlation was found between the parameters studied and either the duration or the level of injury. In conclusion, in contrast to acute SCI, the coagulation profile in uncomplicated chronic SCI is noted to be largely normal with only a few minor alterations of questionable clinical significance.     

Sunlight exposure, hat use, and squamous cell skin cancer on the head and neck

TF (tissue factor) is a physiological inhibitor of blood coagulation in normal hemostasis and is a major initiator of clotting in thrombotic disease. TF functions as a protein cofactor for FVIIa. Coagulation at a site of injury is initiated by exposure of blood to cell-surface formation of TF/VIIa complex. The TF/VIIa complex then activates both factors IX and X leading to thrombin generation and fibrin formation. TFPI (tissue factor pathway inhibitor) appears to play a primary role in regulating TF-induced coagulation. Abnormal coagulation may contribute to the pathogenesis of many serious illnesses. In particular, induced expression of TF and TF-mediated coagulation occurs in atherosclerotic plaques, sepsis, malignancy, ARDS and glomerulonephritis. Several observations support the need for exogenous TFPI administration to effectively turn off the TF/VIIa complex in several clinical conditions with TF-induced coagulopathy. There are some reports about successful administration of rTFPI for antithrombotic therapy in humans.     

Activation-dependent exposure of the inter-EGF sequence Leu83-Leu88 in factor Xa mediates ligand binding to effector cell protease receptor-1

Binding of factor Xa to human umbilical vein endothelial cells (HUVEC) is contributed by effector cell protease receptor-1 (EPR-1). The structural requirements of this recognition were investigated. Factor Xa or catalytically inactive 5-dimethylaminonaphthalene-1sulfonyl (dansyl) Glu-Gly-Arg-(DEGR)-chloromethylketone-factor Xa bound indistinguishably to HUVEC and EPR-1 transfectants, and inhibited equally well the binding of 125I-factor Xa to these cells. Similarly, factor Xa active site inhibitors TAP or NAP5 did not reduce ligand binding to EPR-1. A factor X peptide duplicating the inter-EGF sequence Leu83-Phe84-Thr85-Arg86-Lys87-Leu88- (Gly) inhibited factor V/Va-independent prothrombin activation by HUVEC and blocked binding of 125I-factor Xa to these cells in a dose-dependent manner (IC50 approximately 20-40 microM). In contrast, none of the other factor X peptides tested or a control peptide with the inter-EGF sequence in scrambled order was effective. A recombinant chimeric molecule expressing the factor X sequence Leu83-Leu88 within a factor IX backbone inhibited binding of 125I-factor Xa to HUVEC and EPR-1 transfectants in a dose-dependent fashion, while recombinant factor IX or plasma IXa had no effect. An antibody generated against the factor X peptide 83-88, and designated JC15, inhibited 125I-factor Xa binding to HUVEC. The JC15 antibody bound to factor Xa and the recombinant IX/X83-88 chimera in a concentration dependent manner, while no specific reactivity with factors X or IXa was observed. Furthermore, binding of 125I-factor Xa to immobilized JC15 was inhibited by molar excess of unlabeled factor Xa, but not by comparable concentrations of factors X or IXa. These findings identify the inter-EGF sequence Leu83-Leu88 in factor Xa as a novel recognition site for EPR-1, and suggest its potential role as a protease activation-dependent neo-epitope. This interacting motif may help elucidate the contribution of factor Xa to cellular assembly of coagulation and vascular injury.     

Removal of heparin and protamine from plasma

A simple chromatographic technique for rapid adsorption of heparin and protamine from plasma samples is described, allowing accurate interpretation of coagulation screening tests and specific clotting factor assays. With the use of columns of ECTEOLA-cellulose, up to 300 U. of heparin could be completely adsorbed from a 1 ml. plasma sample. When citrated nonheparinized plasma was passed over the ECTEOLA-cellulose columns, the thrombin, prothrombin, and partial thromboplastin times were unaffected. Levels of fibrinogen, prothrombin, and factors V, VII, VIII, IX, and XI average within 90 per cent of control, nonchromatographed samples. When heparinized plasma samples (0.1 and 1.0 U. per milliliter) were passed over columns, heparin was completely removed and the results of the screening tests and the specific factor assays were the same as for the chromatographed nonheparinized samples. In addition, heparinized samples with decreased factor VIII activity maintained their pretreatment factor VII activities after heparin removal. Blood samples containing heparin were obtained from two patients during open-heart surgery. Following heparin adsorption on ECTEOLA-cellulose columns, factor VIII activity levels remained above 60 per cent during cardiopulmonary by-pass. The presence of protamine sulfate in plasma samples prolonged the prothrombin and partial thromboplastin times while slightly shortening the thrombin time. The protamine effect persisted after ECTEOLA-cellulose, but could be removed by a similar column of carboxymethyl-cellulose. The latter resin had no effect on screening tests or on assays of factors VIII or IX activity. The combination of the two resins was then used to remove the separate inhibitory effects from heparinized plasma samples to which protamine had been added.     

Re-interpreting anaerobic metabolism: an argument for the application of both anaerobic glycolysis and excess post-exercise oxygen comsumption (EPOC) as independent sources of energy expenditure

We studied the effects of porcine factor VIII (P-FVIII; Hyate:C) and other coagulation products employed in the management of patients with hemophilia A, on platelet activation in vitro. Exposure of normal resting platelets to P-FVIII resulted in platelet activation, as manifested by increased expression of the platelet surface activation markers CD62, CD63, and activated-GPIIbIIIa, and by activation-induced modulation of expression of normal platelet membrane glycoproteins CD41, CD42, and CD36. In contrast, platelet activation was not observed after exposure of the platelets to human FVIII, FEIBA, recombinant FVIIa, or cryosupernatant plasma. As with thrombin, exposure of platelets to P-FVIII resulted in the generation of platelet microparticles, an effect not seen not with the other products. In contrast to the characteristic reduction in expression in the number of CD42 molecules detected on thrombin-activated platelets, P-FVIII-stimulated platelets showed a small increase in CD42 expression. In contrast to thrombin, P-FVIII did not cause platelet dense granule release. The results indicate that therapeutic P-FVIII activates platelets, likely in ways that are different from the platelet activation seen with thrombin. The observed platelet activation and microparticle generation may provide a "hypercoagulable" mechanism for hemostasis with P-FVIII therapy separate from, and additional to, that due to increased circulating FVIII levels.     

Dynamic redistribution of glycoprotein Ib/IX on surface-activated platelets. A second look

The present study has re-evaluated the mobility of glycoprotein Ib/IX (GPIb/IX), the von Willebrand factor receptor, on surface-activated platelets. A previous report employing immunogold cytochemistry with monoclonal and polyclonal antibodies specific for GPIb/IX concluded that the receptor remained stabilized in plasma membranes and did not move during platelet attachment and spreading on formvar grids, despite the observation that immunogold particles marking GPIb/IX were missing from peripheral margins and pseudopods of the surface-activated platelets. Addition of thrombin to surface-activated, spread platelets freed GPIb/IX from its anchor to the membrane and stimulated movement of receptor-ligand complexes into caps over centers of spread platelets. In our investigation, surface-activated platelets, stimulated or not by thrombin, were fixed in a higher concentration of glutaraldehyde than used by the earlier workers before exposure to monoclonal or polyclonal antibody to GPIb/IX, after incubation with the antibody, but before treatment with the immunogold marker, protein A gold (PAG), or after both antibody and PAG. When fixed before exposure to antibody and PAG, GPIb/IX receptors were dispersed evenly over dendritic and spread platelets from edge to edge, including peripheral margins and pseudopods. Thrombin had no influence on distribution of the receptors. Exposure to antiglycocalicin antibody before fixation caused movement of GPIb/IX receptors from peripheral margins of spread cells and pseudopods of dendritic forms. Thrombin treatment did not enhance the movement. Fixation after exposure of surface-activated platelets, treated or not with thrombin, to antibody and PAG caused movement of GPIb/IX receptors into caps over cell centers. Results indicate that central movement of GPIb/IX receptors is unrelated to surface activation, spreading, or thrombin stimulation. Rather, the translocation is caused by the antiglycocalicin antibody and accentuated by PAG.     

Procoagulant nature of fibrin

The main threat from a beginning thrombus is that it tends to grow, and hence become occlusive and/or embolise. Although the progressive nature of thrombi has been recognised since a long time, the mechanisms behind thrombus growth remain only partially resolved. In order to investigate in what ways thrombi can themselves become foci of further thrombin -and hence fibrin-formation, we studied the effect of fibrin clots on thrombin generation in platelet poor--and platelet rich plasma (PPP and PRP). The thrombin always adsorbed on a natural fibrin clot is not inactivated by plasmatic antithrombins and could be shown to retain its ability to enhance further thrombin formation by activation of clotting factors V and VIII as well as of blood platelets. To our surprise, fibrin clots without any active thrombin adsorbed, because they were obtained by a snake-venom enzyme or because thrombin had been inhibited, retained their capacity to activate blood platelets and make them procoagulant. The activation could be shown to be due to a rearrangement of cell-membrane phospholipids, by which the procoagulant species (phosphatidyl serine and phosphatidyl ethanolamine) became available at the outer cell surface. The platelet membrane receptor involved could be recognised as glycoprotein Ib, interacting with fibrin through the plasma protein von Willebrand factor (vWf). In fact it appeared that vWf is indispensable for the generation of thrombin in PRP, with or without added clot. This assigns a new and hitherto unknown role to vWf. Our results also show that fibrin is far from being the inert end-product of coagulation but is a potent activator of blood platelets and by this action may foster thrombin generation and hence further fibrin production. We surmise this mechanism to be instrumental in the progression of thrombotic processes.