Analysis of platelet adhesion to a collagen-coated surface under flow conditions: the involvement of glycoprotein VI in the platelet adhesion

Platelet adhesion to the exposed surface of the extracellular matrix in flowing blood is the first and critical reaction for in vivo thrombus formation. However, the mechanism of this in vivo platelet adhesion has yet to be studied extensively. One of the reasons for this is the lack of a practical assay method for assessing platelet adhesion under flow conditions. We have devised an assay method (the fluorescent adhesion assay) that is based on the technique originally reported by Hubbell and McIntire (Biomaterials 7:354, 1986) with some modifications to make it more amenable for assaying small samples and have developed an analysis method to quantify the extent of platelet adhesion and aggregation from fluorescence images by using a computer-assisted image analysis system. In our assay, platelet adhesion, expressed as the percentage of the area covered by adhered platelets, was found to increase biphasically as a function of time. In the first phase, platelets interacted with the coated collagen, transiently stopping on the surface; we called this reaction the temporary arrest. In the second phase, platelets adhered much more rapidly and permanently on the surface, and this adhesion was dependent on the shear rate; platelets formed aggregates in this phase. We used our assay to analyze the effects of platelet aggregation inhibitors on platelet adhesion. All three examined inhibitors, EDTA (10 mmol/L), antiglycoprotein (GP) IIb/IIIa, and GRGDS peptide (1 mmol/L), inhibited the second phase adhesion in flowing blood. Furthermore, GPVI-deficient platelets also showed defective second-phase adhesion under the same conditions. These results suggested that GPIIb/IIIa activation and GPVI contribute to the reaction inducing the second phase. The second-phase adhesion has been extensively investigated, and the consensus is that this reaction is mainly attributable to the platelet-platelet interaction. In this report, we were able to detect an earlier reaction, the temporary arrest. This temporary arrest would reflect the fast and weak interaction between platelet GPIb/IX and collagen-von Willebrand factor complexes on the collagen-coated surface.     

Inhibition of long-chain fatty acid metabolism does not affect platelet aggregation responses

A number of anti-anginal agents (perhexiline, amiodarone, trimetazidine) have been shown to inhibit myocardial carnitine palmitoyltransferase-1, which controls access of long-chain fatty acids to mitochondrial sites of beta-oxidation. In view of clinical data suggesting that perhexiline improves symptomatic status in unstable angina pectoris, and the known role of mitochondrial beta-oxidation in platelet metabolism, we compared the platelet carnitine palmitoyltransferase-1 inhibitory and putative anti-aggregatory effects of perhexiline, amiodarone and trimetazidine with those of specific carnitine palmitoyltransferase-1 inhibitors: etomoxir and hydroxyphenylglyoxylate in both normal subjects and patients with stable angina. All of the compounds examined inhibited platelet carnitine palmitoyltransferase-1 activity; rank order of potency etomoxir > malonyl-CoA > hydroxyphenylglyoxylate > amiodarone > or = perhexiline > trimetazidine. However, only perhexiline, amiodarone and trimetazidine inhibited platelet aggregation. We conclude that (a) the carnitine palmitoyltransferase-1 inhibitors perhexiline, amiodarone and trimetazidine exert significant anti-aggregatory effects which may be therapeutically relevant and, (b) these effects are independent of carnitine palmitoyltransferase-1 inhibition.     

Polymorphisms of platelet membrane glycoprotein Ib associated with arterial thrombotic disease

Platelet membrane glycoprotein Ibalpha (GPIbalpha) is a major receptor for von Willebrand factor and thrombin, which plays a key role in the initial development of thrombi. Two polymorphisms (HPA-2 and VNTR) that affect phenotype have been described in GPIbalpha. The relevance of these polymorphisms to thrombotic disease was investigated by genotypic identification in three case-control studies: 104 case patients with acute cerebrovascular disease (CVD), 101 case patients with acute coronary heart disease (CHD), 95 patients with deep venous thrombosis (DVT), and one control age-, sex-, and race-matched for each case patient. Results show that the C/B genotype of the VNTR and the HPA-2b polymorphisms of GPIbalpha are strongly associated with increased risk of coronary heart disease and cerebral vascular disease but not with deep vein thrombosis. These two polymorphisms of GPIbalpha may represent newly identified risk factors for arterial thrombotic disease, but not for venous thrombosis.     

Platelet adhesion to collagen type IV under flow conditions

Collagen type IV is a sheet-forming collagen and a major constituent of the vessel wall. To find out which conditions are important for platelet adhesion to collagen type IV, we performed perfusion studies with anticoagulated blood in parallel plate perfusion chambers. The role of divalent cations was investigated by using plasmas with variable concentrations of Mg2+ and Ca2+ ions. When Mg2+ concentration was decreased from 2.00 mmol/L to 0.25 mmol/L at a fixed Ca2+ concentration of 1.25 mmol/L, platelet coverage on the collagen type IV surface decreased from 22.8% +/- 1.8% (n = 4) to 4.6% +/- 0.6% (n = 4) at a shear rate of 1,600 s-1. Also, platelet aggregate formation on collagen type IV was strongly impaired. A monoclonal antibody against the glycoprotein (Gp) Ib receptor and von Willebrand factor (vWF)-depleted plasma reduced the platelet coverage to collagen type IV to, respectively, 10% and 45% of the control value. Electron microscopy showed that vWF was only present between platelets and between the platelet and the collagen type IV surface, but did not bind elsewhere to collagen type IV. These data indicate that collagen type IV is a reactive collagen for platelets. Differences in physiologic plasma magnesium concentrations may in part explain the differences in platelet reactivity to collagen type IV between individuals, and perhaps contribute to differences in the risk for thrombosis.     

Differences between platelet phosphoinositide metabolism stimulated by thrombin or SFLLRN are not accounted for by interaction of thrombin with glycoprotein Ib

Tissues from 95 bottlenose dolphins (Tursiops truncatus) that died during the 1987-1988 US Atlantic coast epizootic and 11 bottlenose dolphins that died along the Atlantic coast prior to 1987 were examined histologically and immunohistochemically. Polymerase chain reaction (PCR) testing was performed on 36 of the epizootic and all of the pre-1987 cases. Epizootic cases had syncytia and rare intranuclear and intracytoplasmic inclusion bodies within lung, lymph node, and spleen. Lymphoid depletion was present in lymph node, spleen, and gut-associated lymphoid tissue of epizootic cases. Pre-1987 cases did not have these pulmonary and lymphoid lesions. A larger percentage of epizootic than pre-1987 cases had bacterial and/or fungal infections (primarily pneumonias), pulmonary and lymphoid tissue histiocytosis, mucocutaneous ulcers, and evidence of negative energy balance. Immunohistochemically, 49/95 (52%) epizootic dolphins were positive for morbilliviral antigen. Morbilliviral antigen was detected in lung, lymph node, spleen, thymus, skin, tongue, esophagus, liver, pancreas, gastrointestinal tract, urinary bladder, oviduct, and mammary gland by immunohistochemistry. PCR testing identified morbilliviral RNA in 35/36 (97%) epizootic cases tested. Neither morbilliviral antigen nor morbilliviral RNA were detected in pre-1987 cases. Histologic, immunohistochemical, and PCR results provide strong evidence that morbillivirus infection was the primary cause of the 1987-1988 bottlenose dolphin epizootic.     

Thrombin promotes platelet-mediated melanoma cell adhesion to endothelial cells under flow conditions: role of platelet glycoproteins P-selectin and GPIIb-IIIA

We investigated the role of platelets in human melanoma cell (line 397) interaction with vascular endothelial cells (ECs) under flow conditions. The ability of the tumour cells to adhere to the EC monolayer was significantly reduced by application of flow at a shear rate of 250 s(-1). A 2.2-fold increase in tumour cell adhesion to ECs under flow was observed upon addition of thrombin receptor agonist peptide (TRAP)-activated platelets but not resting platelets. A similar increase (2.5-fold) in tumour cell adhesion to ECs under flow was observed when the tumour cells were incubated with resting platelets on thrombin-treated ECs. However, thrombin treatment of the ECs alone had no effect on tumour cell adhesion in the absence of platelets. The enhancement of tumour cell adhesion to ECs by TRAP-activated platelets was virtually abolished by blockade of the platelet glycoproteins P-selectin and GPIIb-IIIa by monoclonal antibodies. Blockade of P-selectin also inhibited the direct adhesion of TRAP-activated platelets to ECs, but did not affect the interaction of the tumour cells with platelets immobilized on subendothelial extracellular matrix (ECM). Blockade of GPIIb-IIIa inhibited both platelet-EC and platelet-tumor cell interactions. Our results indicate that tumour cell adhesion to the endothelium under flow is enhanced by platelets under conditions that allow platelet adhesion to ECs. Inhibition studies suggest that activated platelet adhesion to ECs is mediated by P-selectin and GPIIb-IIIA, and tumour cell adhesion to EC-bound platelets--mainly by GPIIb-IIIa.     

Eprosartan does not affect the pharmacodynamics of warfarin

The purpose of this study was to conduct a retrospective analysis of the clinical spectrum, treatment and morbidity of the patients who have suffered high tension electrical injuries with current passage through their body (59 patients). Voltage, localization and surgical treatment seem to be the main factors influencing the lesion and the morbidity. The following points were considered: (1) Is there any relation between known factors such as voltage and the localization of the points of contact with the incidence and the type of complications and sequelae? (2) Do the observations show that wound management and the excision of dead tissues is the most adequate? From factors studied in our patients (voltage, point of entry and pathway of current, associated multiple trauma or flame burns, surgical treatment) we have found that the voltage does not have any influence on the severity of the wound nor on the percentage of sequelae (cataracts, limb amputation, neurologic complications). The current pathway, as well as its points of entry, does not show any relation with the presence of renal failure, cardiac arrhythmia and cataracts. A clear relationship between the point of entry of the current and the appearance of neurologic injury with presence of paralysis and permanent regional anaesthesia at the same level was observed. The presence of associated burns was not related to any other complications or sequelae. For those patients whose length of contact has been shorter we find a lower rate of amputations despite having associated limb fractures. Fasciotomy incisions appear to confer benefit as this series shows that this procedure decreases the rate of limb amputations.     

A role for von Willebrand factor proline residues 702-704 in ristocetin-mediated binding to platelet glycoprotein Ib

Mutant domains of von Willebrand factor (vWF) were constructed to determine the effects of altering net charge, and presumably conformation, within a peptide sequence (residues 694-708) previously shown to be involved in the platelet receptor glycoprotein (GP) Ib binding function of vWF. Non-conservative substitutions replaced a triplet of proline residues (proline702-704) with either a triplet of arginine (positively-charged) or aspartic acid (negatively-charged) residues. After establishing stable CHO cell transformants, we observed the secretion of covalently-linked dimeric molecules analogous to a domain with native sequence. Functional assays using immunopurified molecules revealed that the ristocetin-dependent binding to GP Ib was abolished with both charge mutants. However, in the absence of disulfide-bond dependent conformation both mutant molecules and the molecule with native sequence interacted with GP Ib. The results demonstrate that vWF proline702-704 are important for the ristocetin-mediated interaction between vWF and GP Ib, but are not essential residues of the GP Ib binding site within vWF.     

A new model to assess staphylococcal adhesion to intraocular lenses under in vitro flow conditions

Adhesion of staphylococcal cells to intraocular lenses coated with heparin was studied under in vitro flow conditions (280 microl min(-1)) at 37 degrees C. The intraocular lenses were incubated with human cerebrospinal fluid for 1 h or with cerebrospinal fluid including 0.50% plasma for 12 h, prior to bacterial challenge. Two strains of Staphylococcus epidermidis selected for this study, were isolated from biomaterial-associated infections. Bacterial adhesion was quantitated by bioluminescence and visualized by fluorescence microscopy of acridine orange stained bacteria. Surface coating with heparin significantly decreased bacterial adhesion of both strains after incubation with cerebrospinal fluid including 0.50% plasma for 12 h (p = 0.0209). However, no difference in bacterial adhesion was obtained between intraocular lenses with and without heparin, after incubation with cerebrospinal fluid for 1 h (p = 0.327). Microscopy showed that more bacteria were present on intraocular lenses without heparin than on intraocular lenses with heparin. The results show that preincubation with a proteinaceous fluid influences subsequent bacterial adhesion to the polymer surface. The results suggest that IOLs with heparin coating may be less prone to bacterial adhesion under perfusion conditions after surface conditioning in human CSF with 0.50% plasma and a preincubation period of 12 h. Heparin coating might be a valuable tool to decrease implant-associated bacterial endophthalmitis.     

Antithrombotic effect of crotalin, a platelet membrane glycoprotein Ib antagonist from venom of Crotalus atrox

A potent platelet glycoprotein Ib (GPIb) antagonist, crotalin, with a molecular weight of 30 kD was purified from the snake venom of Crotalus atrox. Crotalin specifically and dose dependently inhibited aggregation of human washed platelets induced by ristocetin with IC50 of 2.4 microg/mL (83 nmol/L). It was also active in inhibiting ristocetin-induced platelet aggregation of platelet-rich plasma (IC50, 6.3 microg/mL). 125I-crotalin bound to human platelets in a saturable and dose-dependent manner with a kd value of 3.2 +/- 0.1 x 10(-7) mol/L, and its binding site was estimated to be 58,632 +/- 3, 152 per platelet. Its binding was specifically inhibited by a monoclonal antibody, AP1 raised against platelet GPIb. Crotalin significantly prolonged the latent period in triggering platelet aggregation caused by low concentration of thrombin (0.03 U/mL), and inhibited thromboxane B2 formation of platelets stimulated either by ristocetin plus von Willebrand factor (vWF), or by thrombin (0.03 U/mL). When crotalin was intravenously (IV) administered to mice at 100 to 300 microg/kg, a dose-dependent prolongation on tail bleeding time was observed. The duration of crotalin in prolonging tail bleeding time lasted for 4 hours as crotalin was given at 300 microg/kg. In addition, its in vivo antithrombotic activity was evidenced by prolonging the latent period in inducing platelet-rich thrombus formation by irradiating the mesenteric venules of the fluorescein sodium-treated mice. When administered IV at 100 to 300 microg/kg, crotalin dose dependently prolonged the time lapse in inducing platelet-rich thrombus formation. In conclusion, crotalin specifically inhibited vWF-induced platelet agglutination in the presence of ristocetin because crotalin selectively bound to platelet surface receptor-glycoprotein Ib, resulting in the blockade of the interaction of vWF with platelet membrane GPIb. In addition, crotalin is a potent antithrombotic agent because it pronouncedly blocked platelet plug formation in vivo.     

Promiscuity of clinical Plasmodium falciparum isolates for multiple adhesion molecules under flow conditions

The central pathologic process in severe Plasmodium falciparum malaria is the cytoadherence of parasitized erythrocytes to capillary and postcapillary venular endothelium, with resultant tissue hypoxia, metabolic disturbances, and multiorgan dysfunction. The molecular basis of this process has been studied extensively using static adhesion assays. In the present study, we determined whether infected red blood cells (IRBC) from clinical parasite isolates would roll and adhere on CD36, ICAM-1, E-selectin, P-selectin, and VCAM-1 using a laminar flow system that allowed for the direct visualization of IRBC-substratum interactions. The results indicate that IRBC could tether and roll on CD36, ICAM-1, P-selectin, and VCAM-1 in a shear-dependent fashion, but significant adhesion was restricted to CD36. There was no interaction with E-selectin. When both CD36 and ICAM-1 were expressed on the same cellular substratum such as C32 melanoma cells, adhesion was significantly greater than when CD36 was present alone. The adhesive interactions were different from those between leukocytes and the same adhesion molecules. Furthermore, IRBC rolling on P-selectin and VCAM-1 was not inhibitable by Abs that entirely prevented leukocyte-receptor interactions. These findings suggest that cytoadherence under physiologic conditions may be a multistep process similar to that involved in the recruitment of a number of different cell types. Further elucidation of the molecular basis of these novel interactions is crucial for the development of therapeutic interventions aimed at inhibiting or reversing the process.     

Rhodocytin, a functional novel platelet agonist belonging to the heterodimeric C-type lectin family, induces platelet aggregation independently of glycoprotein Ib

We isolated and characterized a functionally novel platelet agonist, designated as rhodocytin, from the Calloselasma rhodostoma venom. Rhodocytin was a disulfide-linked heterodimer consisting of 18- and 15-kDa subunits. The respective N-terminal amino acid sequences of both subunits were homologous to each other and to those of the carbohydrate-recognition domains (CRD) of C-type lectins. Rhodocytin alone induced platelet aggregation. Platelet agonists and antagonists constructed with CRD-like subunits from snake venoms bind to glycoprotein Ib directly or indirectly. However, rhodocytin induced platelet aggregation not by binding to glycoprotein Ib, because rhodocytin-induced platelet aggregation was not influenced by echicetin, a glycoprotein Ib-binding protein, that completely inhibits platelet agglutination by bovine von Willebrand factor. These findings indicate that rhodocytin is a novel protein structurally related to heterodimers of CRD-like subunits, but functionally distinct from venom proteins that induce platelet aggregation via glycoprotein Ib.     

Prelabeled glycoprotein Ib/IX receptors are not cleared from exposed surfaces of thrombin-activated platelets

The present investigation has re-examined the hypothesis proposing that glycoprotein (GP)Ib/IX receptors for von Willebrand factor are rapidly cleared from exposed surfaces to internal membrane systems after activation of platelets by thrombin in suspension. Platelets were prelabeled with either a polyclonal antibody to GPIb alpha, antiglycocalicin (A-Gl), or a cocktail of two monoclonal antibodies, AP1 and 6D1, exposed to 0.1 or 0.2 U/ml thrombin for 5 or 10 minutes, fixed and stained with Staphylococcus protein A coupled to gold to detect A-Gl or goat anti-mouse IgG bound to gold particles to locate AP1 and 6D1 before or after preparation of frozen thin sections or embedding for plastic thin sections. The frequency and distribution of protein-A-gold markers for GPIb/IX on thrombin-activated platelets viewed in thin plastic sections did not differ from the density on resting platelets stained with A-Gl. Cryosections of A-Gl-prelabeled platelets labeled again on cryosections revealed GPIb present on linings of the open canalicular system of resting and activated platelets, but the density of gold in interior channels and frequency of gold particles on exterior surfaces were not altered by thrombin stimulation. Platelets prelabeled with the cocktail of 6D1 and AP1 and studied in cryosections also failed to reveal uptake of GPIb/IX receptors into the open canalicular system after activation by thrombin. The findings do not support the concept that thrombin causes clearance of GPIb/IX receptors from exterior surfaces to interior membranes of activated platelets.     

IL-12 promotes the adhesion of NK cells to endothelial selectins under flow conditions

This study examined the adhesive interactions of peripheral blood NK cells with P- and E-selectin and analyzed the effect of IL-12 on the binding of NK cells to these selectins. P-selectin glycoprotein ligand-1 (PSGL-1) is expressed on most resting and IL-12-activated NK cells. However, the percentage of resting NK cells bound to P-selectin-IgG was 15%, and that of activated NK cells bound to P-selectin-IgG was 65%. Furthermore, the number of IL-12-activated NK cells bound to P-selectin-transfected Chinese hamster ovary cells was significantly higher than that of resting NK cells under flow conditions. These interactions were abolished by the incubation of these NK cells with anti-PSGL-1 (PL-1) mAb. Thus, PSGL-1/P-selectin interaction is important in the binding of resting and activated NK cells to P-selectin. NK cells express sialyl-Lewis(x) (sLe(x)) structure recognized by anti-sLe(x) mAb (KM-93), and IL-12 activation of NK cells increased the mean fluorescence intensity of KM-93-reactive NK cells. Adhesion of IL-12-activated NK cells to E-selectin-transfected Chinese hamster ovary cells was stronger than that of resting NK cells under flow conditions. These interactions were reduced markedly by incubation with anti-sLe(x) mAb. Thus, sLe(x) is the major ligand of resting and activated NK cells for E-selectin. These findings indicate that IL-12 stimulation of NK cells promotes their adhesion activity to endothelial selectins.     

High D-glucose does not affect binding of alpha-tocopherol to human erythrocytes

The role of alpha-tocopherol uptake system in human erythrocyte in the uptake of plasma alpha-tocopherol has been suggested. However no information is available on alpha-tocopherol uptake activity of human erythrocytes in the presence of high levels of D-glucose which is known to lead to pathological alterations in different cells including human erythrocytes. Therefore, in order to examine the effect of D-glucose on the binding of alpha-tocopherol to human erythrocytes, the binding characteristics of alpha-tocopherol to these cells were established first. Binding of [3H]alpha-tocopherol to human erythrocytes was both saturable and specific. Scatchard analysis of alpha-tocopherol binding to these cells showed the presence of two independent classes of binding sites with widely different affinities. The high affinity binding sites had a dissociation constant (Kd1) of 90 nM with a binding capacity (n1) of 900 sites per cell, whereas the low affinity binding sites had a dissociation constant (Kd2) of 5.2 microM and a binding capacity (n2) of 105,400 sites per cell. Trypsin treatment abolished all the alpha-tocopherol binding activity. Competition for the binding of alpha-tocopherol to human erythrocytes was effective with other homologues of alpha-tocopherol (beta-tocopherol, gamma-tocopherol and delta-tocopherol) and their potency was almost equal to alpha-tocopherol itself. The order of preference was alpha-tocopherol > beta-tocopherol > or = gamma-tocopherol > or = delta-tocopherol. Incubation of human erythrocytes with various concentrations of D-glucose did not affect alpha-tocopherol uptake activity. Our data demonstrate the presence of an alpha-tocopherol uptake system in human erythrocytes and that the alpha-tocopherol uptake activity is not modulated by the presence of D-glucose.     

Depletion of striatal dopamine transporter does not affect psychostimulant-induced locomotor activity

The effect of neurotoxin-induced depletion of striatal dopamine transporter (DAT) binding sites on animals' responses to psychostimulants was investigated. Multiple 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or methamphetamine (METH) injections but not a single METH injection to Swiss Webster mice resulted in > 60% depletion of striatal DAT. MPTP-induced depletion of DAT did not affect METH- and cocaine-stimulated locomotor activity compared with the response of control mice. Pre-exposure to either the neurotoxic or the single non-neurotoxic dose of METH resulted in a marked locomotor sensitization in response to METH or cocaine challenge injections. The present results indicate that > 60% loss in striatal DAT binding sites has no effect on animals' responses to psychostimulants, and suggest that neural systems other than striatal DAT may contribute to the induction of locomotor sensitization to METH and cocaine.     

Zinc status does not affect aluminum deposition in tissues of rats

To examine whether zinc deficiency would increase the toxicity of dietary aluminum, weanling, male Sprague-Dawley rats were fed purified diets containing either 2 or 30 mg Zn/kg diet, with or without 500 mg Al/kg diet for 28 d. Individually pair-fed rats were fed the 30 mg Zn/kg diet with or without added aluminum to control for inanition secondary to zinc deficiency. Rats fed the 2 micrograms Zn/kg diet showed evidence of zinc deficiency, including anorexia, growth retardation, and depressed concentrations of zinc in tibias and livers. Zinc deficiency did not significantly increase the concentrations of aluminum in the tibias, livers, kidneys, or regions of the brain examined (cerebrum, cerebellum, midbrain, and hippocampus). Inclusion of aluminum in the diet did not alter aluminum concentrations in the various tissues. Under the conditions of this study, zinc deficiency did not result in greater sensitivity to dietary aluminum exposure.     

Inhibition of neutrophil adhesion does not prevent ischemic spinal cord injury

Paraplegia may occur after transient aortic occlusion as a consequence of primary ischemia to the spinal cord or injury during the reperfusion period. In animal models of ischemia/reperfusion there is evidence that reperfusion injury may be modulated partially by neutrophils. The efficacy of the neutrophil adherence blocking murine monoclonal antibody (MAb 60.3) was assessed in spinal cord ischemia/reperfusion in rabbits. Spinal cord ischemia was accomplished by balloon catheter occlusion of the infrarenal aorta. Neurologic assessment was graded as normal, partial neurologic deficit, or complete paralysis. Electrophysiologic monitoring with somatosensory evoked potentials was used to determine the optimal length of time of occlusion. Animals were treated randomly with 2 mg/kg of intravenous Mab 60.3 (n = 8) or saline solution (n = 9) with the investigator unaware of treatment. Mean occlusion times were no different between groups (control, 32.7 +/- 3.6 minutes versus MAb, 32.4 +/- 6.0 minutes). Five (55%) saline-treated and four (50%) MAb 60.3-treated animals became paraplegic. Animals with initial paraparesis all progressed to flaccid paraplegia within 24 hours. We conclude that spinal cord injury after transient aortic occlusion is independent of the CD11/CD18 glycoprotein complex of the neutrophil. Injury in this setting may occur during ischemia and thus may not be dependent on neutrophils or reperfusion.