On the role of von Willebrand factor in promoting platelet adhesion to fibrin in flowing blood

Platelet adhesion to fibrin at high shear rates depends on both the glycoprotein (GP) IIb:IIIa complex and a secondary interaction between GPIb and von Willebrand factor (vWF). This alternative link between platelets and vWF in promoting platelet adhesion to fibrin has been examined in flowing whole blood with a rectangular perfusion chamber. Optimal adhesion required both platelets and vWF, as shown by the following observations. No binding of vWF could be detected when plasma was perfused over a fibrin surface or when coated fibrinogen was incubated with control plasma in an enzyme-linked immunosorbent assay. However, when platelets were present during perfusion, interactions between vWF and fibrin could be visualized with immunoelectron microscopy. Exposure of fibrin surfaces to normal plasma before perfusion with severe von Willebrand's disease blood did not compensate for the presence of plasma vWF necessary for adhesion. vWF mutants in which the GPIIb:IIIa binding site was mutated or the GPIb binding site was deleted showed that vWF only interacts with GPIb on platelets in supporting adhesion to fibrin and not with GPIIb:IIIa. Complementary results were obtained with specific monoclonal antibodies against vWF. Thus, vWF must first bind to platelets before it can interact with fibrin and promote platelet adhesion. Furthermore, only GPIb, but not GPIIb:IIIa is directly involved in this interaction of vWF with platelets.     

The role of platelet membrane glycoproteins Ib and IIb-IIIa in platelet adherence to human artery subendothelium

Platelet adherence to human artery subendothelium in blood from eight normal subjects, four patients with Glanzmann's thrombasthenia (deficiency of platelet membrane glycoproteins IIb and IIIa: GPIIb-IIIa), two patients with Bernard-Soulier syndrome (deficiency of platelet membrane glycoprotein Ib: GPIb) and one patient with von Willebrand's disease (VWD subtype III. deficient in factor VIII-von Willebrand factor: FVIII-VWF) was compared at various wall shear rates (300, 500, 1000, 1800 and 2500 s-1). Platelet adherence in blood from the patients with Glanzmann's thrombasthenia was within the normal range at shear rates below 1000 s-1. There was some decrease in adhesion at higher shear rates and platelets were less spread out on the subendothelium than normally at all shear rates. Platelet aggregate formation was almost totally absent. Platelet adherence in blood from patients with the Bernard-Soulier syndrome was strongly impaired at all shear rates. Platelet adherence in blood from the patient with VWD subtype III was normal at shear rates of 300 and 500 s-1, but impaired at shear rates above 1000 s-1. Aggregate formation was also decreased at these shear rates. Platelet adhesion was strongly inhibited by a monoclonal antibody against glycoprotein Ib, which had previously been shown to inhibit ristocetin-induced aggregation, at shear rates of 500 and 1800 s-1 but not at 300 s-1. Platelet adhesion at 1800 s-1 was also inhibited, though to a lesser extent, by two antibodies against GPIIb-IIIa. These antibodies also inhibited platelet aggregate formation. The data indicates that GPIb is involved in adhesion at the same shear rates as von Willebrand factor. Absence or inhibition of GPIIb-IIIa primarily causes a defect of aggregate formation but GPIIb-IIIa may also play a role in adhesion, particularly at high shear rates. The defect of adhesion in the Bernard-Soulier syndrome may be dependent on factors other than a deficiency of GPIb alone.     

Characterization of murine anti-glycoprotein Ib monoclonal antibodies that differentiate between shear-induced and ristocetin/botrocetin-induced glycoprotein Ib-von Willebrand factor interaction

Platelet adhesion to vascular subendothelium under conditions of high shear stress is mediated by the platelet glycoprotein (GP) Ib-von Willebrand Factor (vWF) interaction. The aim of this study was to characterize the murine monoclonal antibodies (MoAbs) 27A10 and 28E6, both raised against purified GPIb. The MoAb 27A10 is a potent inhibitor of shear-induced platelet adhesion to collagen type I in a flow chamber at shear rates of 1,300 and 2,700 s(-1). 20 microg/ml of MoAb 27A10, furthermore, could completely block shear-induced aggregation in a modified Couette viscometer at shear rates of 1,000 and 4,000 s(-1). On the other hand, MoAb 27A10 had a negligible effect on botrocetin-induced GPIb-vWF binding and is only a poor inhibitor of the ristocetin-dependent interaction. In contrast, MoAb 28E6 did abolish both the ristocetin- and botrocetin-induced GPIb-vWF binding, whereas it did not block the shear-induced interaction. Thus, we identify here two anti-GPIb MoAbs 27A10 and 28E6 that either preferentially inhibit the shear-induced or the ristocetin/botrocetin-induced platelet-vWF interaction. With these tools it should be possible to more clearly define the mechanisms by which platelets bind to vWF in vivo.     

Platelet adhesion and aggregation on human type VI collagen surfaces under physiological flow conditions

Type VI collagen is a subendothelial constituent that binds von Willebrand factor (vWF) and platelets. The interaction of platelets with type VI collagen and the roles of platelet glycoprotein (GP) receptors and vWF were studied under flow conditions using epi- fluorescent videomicroscopy coupled with digital image processing. We found that surface coverage was less than 6% on collagen VI at a relatively high-wall shear rate (1,000 s-1) and was approximately 60% at a low-wall shear rate (100 s-1). The molecular mechanisms involved in low-shear platelet binding were studied using monoclonal antibodies to platelet GPIb and GPIIb-IIIa, and polymeric aurin tricarboxylic acid. Anti-GPIIb-IIIa was the most effective in eliminating adhesion (surface coverage, 0.8%), followed by anti-GPIb (4.3%), and ATA (12.6%). Experiments with von Willebrand disease blood indicate that vWF is involved in platelet adhesion to collagen VI at 100 s-1. In the absence of vWF, there may be direct binding of platelet GPIIb-IIIa complexes to collagen VI. Adhesion and aggregation on collagen VI are different in shear rate dependence from collagen I. Our results suggest a possible role for collagen VI and vWF in platelet adhesion and aggregation in vascular regions with low shear rates.     

von Willebrand factor competes with fibrin for occupancy of GPIIb:IIIa on thrombin-stimulated platelets

We have investigated two major questions related to the molecular basis of interactions between the three-dimensional fibrin network and thrombin-stimulated human platelets. First, what are the roles played by glycoproteins (GP) Ib and IIb:IIIa in linking the fibrin clot tightly to the platelet surface? Second, does von Willebrand factor (vWF) modulate the extent of platelet-fibrin interactions? Quantitative fluorescence microscopy (microfluorimetry) has been used to determine the quantity of fluorescein-labeled fibrin bound to the surface of thrombin-stimulated, gel-filtered platelets (the supernatants of which contained small quantities of vWF) in the presence/absence of receptor-specific and vWF-specific monoclonal antibodies (MoAbs), as well as exogenous vWF. A MoAb specific for the GPIIb:IIIa complex exhibited a concentration-dependent inhibition of fibrin binding, whereas a MoAb specific for GPIb was ineffective in this regard. Similarly, a MoAb that recognizes the N-terminal region of vWF involved in GPIb binding did not influence fibrin binding. In contrast, a MoAb that binds to a C-terminal region of vWF involved in GPIIb:IIIa recognition caused a specific, concentration-dependent increase in the quantity of platelet-bound fibrin. We also found that exogenous vWF caused a concentration-dependent decrease in fibrin binding. These results support the hypothesis that vWF and fibrin, both of which are multimeric adhesive ligands, compete for occupancy of the GPIIb:IIIa complex on thrombin-stimulated platelets.     

Fibronectin is required for platelet adhesion and for thrombus formation on subendothelium and collagen surfaces

Fibronectin (FN) plays a role in several adhesion mediated functions including the interaction of platelets with subendothelium. We investigated the role of plasma FN in platelet adhesion and platelet thrombus formation under flow conditions. We used two different perfusion models: the annular chamber with alpha-chymotrypsin-treated rabbit vessel segments, and the flat chamber with coverslips coated with fibrillar purified human collagen type III. Perfusates consisted of washed platelets and washed RBCs, suspended in normal or FN-depleted plasma. Perfusions were carried out for ten minutes at shear rates of 300 or 1,300 s-1. Platelet deposition and thrombus dimensions were evaluated morphometrically by a computerized system. We found that depletion of plasma fibronectin significantly reduced the percentage of total coverage surface and percentage of platelet thrombus, at both shear rates studied, and in both perfusion systems (P less than .01) (P less than .01). The dimensions of the platelet thrombi formed in perfusions at high shear rate were also significantly reduced in perfusions carried out with FN depleted plasma (P less than .01). Addition of purified FN to FN-depleted perfusates restored all values to those measured in the control perfusions. These results indicate that plasma FN is required for platelet aggregate and thrombus formation following adhesion under flow conditions.     

Von Willebrand factor in the vessel wall mediates platelet adherence

A monoclonal antibody directed against the von Willebrand factor moiety (vWF) of factor VIII-von Willebrand factor (FVIII-vWF), which blocks ristocetin-induced platelet aggregation as well as the binding of FVIII- vWF to platelets in the presence of ristocetin, inhibited platelet adherence to human artery subendothelium when present in normal flowing blood. This monoclonal antibody, CLB-RAg 35, inhibited platelet adherence as a function of the shear rate. At wall shear rates below 500 s-1, platelet adherence was not affected, but at higher shear rates platelet adherence was gradually inhibited, reaching an average of 11% of the normal value at 2,500 s-1. Indirect immunofluorescence established the reactivity of CLB-RAg 35 with vWF present in artery subendothelium. Pretreatment of normal vessel walls with this antibody inhibited adherence of platelets in blood from a patient with severe homozygous von Willebrand's disease and in blood from normal individuals. The inhibition was shear-rate dependent and significant at high shear rates (2,500 s-1). By adding increasing amounts of purified FVIII-vWF to normal blood, the inhibition was gradually overcome. These data indicate that vWF present in the vessel wall contributes appreciably to platelet adherence. At high wall shear rates, platelet adherence is mediated virtually completely by both plasma FVIII-vWF and vWF in the vessel wall. At low wall shear rates (below 500 s-1), platelet adherence occurs independent of FVIII-vWF in plasma and vWF in the vessel wall.     

Mechanisms of platelet aggregation by Streptococcus sanguis, a causative organism in infective endocarditis

Summary. The ability of certain strains of Streptococcus sanguis to aggregate human platelets in vitro may be related to their virulence in the pathogenesis of infective endocarditis. We have studied the mechanisms of aggregation of human platelets by S. sanguis strain NCTC 7863. Platelet aggregation follows incubation of S. sanguis cells with platelet-rich plasma from normal, healthy adults, after a lag of 7–19 min. Platelet aggregation was accompanied by 5-hydroxytryptamine release and thromboxane B₂ production. Aggregation was prevented by aspirin and by EDTA. Platelets from two patients with Glanzmann's thrombasthenia did not respond to bacteria. Fixed, washed platelets resuspended in normal plasma were not agglutinated by S. sanguis.
Blocking the glycoprotein Ib receptor with a monoclonal antibody inhibited aggregation of PRP. However, S. sanguis did not induce von Willebrand factor (vWF) binding to platelets; nor did the bacteria prevent ristocetin-induced platelet agglutination or vWF binding. The aggregation response was not related to plasma vWF activity levels in normal subjects or in patients with von Willebrand's disease.
The platelet response to S. sanguis therefore resembles true aggregation, requiring the cyclo-oxygenase pathway and the presence of glycoprotein IIb/IIIa. The mechanism also involves glycoprotein Ib, but not apparently through irreversible binding of vWF.     

Comparative antiplatelet efficacy of a novel, nonpeptide GPIIb/IIIa antagonist (XV454) and abciximab (c7E3) in flow models of thrombosis

Glycoprotein (GP) IIb/IIIa is pivotal in homotypic platelet aggregation and may also be involved in the heterotypic adhesion of leukocytes and tumor cells to platelets. This study was primarily undertaken to compare the antiplatelet efficacy of a novel, nonpeptide GPIIb/IIIa antagonist, XV454, to that of abciximab in 2 flow models of platelet thrombus formation: (1) direct shear-induced platelet aggregation imposed by a cone-and-plate rheometer and (2) platelet adhesion onto von Willebrand factor (vWF)/collagen I followed by aggregation in a perfusion system. XV454 inhibited platelet aggregation in a concentration-dependent manner in both experimental models. Maximal inhibition of aggregation was achieved by XV454 at approximately 70% receptor occupancy, which is lower than the >/=85% previously reported for abciximab. At similar levels of receptor blockade (approximately 45%), XV454 appeared to be relatively more effective than abciximab in suppressing platelet aggregation. Neither XV454 nor abciximab inhibited platelet adhesion to collagen. Pretreatment of surface-adherent platelets with either XV454 or abciximab inhibited the attachment of monocytic THP-1 cells under flow. In contrast, the rapidly reversible GPIIb/IIIa inhibitor orbofiban failed to suppress these heterotypic interactions. These findings demonstrate that XV454 is a potent GPIIb/IIIa antagonist with a long receptor-bound lifetime like abciximab and may be beneficial for the treatment/prevention of thrombotic complications.     

Platelet von Willebrand factor: evidence for its involvement in platelet adhesion to collagen

Although it is well established that plasma von Willebrand Factor (vWF) is essential to platelet adhesion to subendothelium at high shear rates, the role of platelet vWF is less clear. We studied the respective role of both plasma and platelet vWF in mediating platelet adhesion to fibrillar collagen in a parallel-plate perfusion chamber. Reconstituted blood containing RBCs, various mixtures of labeled washed platelets and plasma from controls or five patients with severe von Willebrand disease (vWD), was perfused through the chamber for five minutes at a shear rate of 1,600 s-1. Platelet-collagen interactions were estimated by counting the radioactivity in deposited platelets and by quantitative morphometry. When the perfusate consisted of normal platelets suspended in normal plasma, platelet deposition on the collagen was 24.7 +/- 3.6 X 10(6)/cm2 (mean +/- SEM, n = 6). Significantly less deposition (16 +/- 2.3) was observed when vWD platelets were substituted for normal platelets. In mixtures containing vWD plasma, significantly greater deposition (9 +/- 2.2) was obtained with normal than with vWD platelets (1 +/- 0.4) demonstrating a role for platelet vWF in mediating the deposition of platelets on collagen. Morphometric analysis confirmed these data. Our findings indicate that platelet, as well as plasma, vWF mediates platelet-collagen interactions at a high shear rate.     

Relative antithrombotic effects of monoclonal antibodies targeting different platelet glycoprotein-adhesive molecule interactions in nonhuman primates

The relative antithrombotic effectiveness of targeting glycoprotein (GP) Ib-dependent versus GPIIb-IIIa-dependent platelet interactions has been determined in baboons by measuring thrombus formation after infusing comparable antihemostatic doses of anti-von Willebrand factor (vWF) monoclonal antibody (MoAb) BB3-BD5, anti-GPIb MoAb AP1, and anti- GPIIb-IIIa MoAb LJ-CP8 under conditions of arterial and venous flow (shear rates of 750 to 1,000 seconds-1 and 100 seconds-1, respectively). Thrombus formation was quantified as 111In-platelet deposition and 125I-fibrin accumulation on segments of collagen-coated tubing interposed in chronic exteriorized arteriovenous (AV) shunts for 40 minutes. In vitro, anti-vWF MoAb BB3 BD5 (IgG) and anti-GPIb MoAb AP1 [IgG or F(ab)2 fragments] inhibited ristocetin-induced platelet aggregation (IC50 50 nmol/L and 1 mumol/L, respectively), but neither of these MoAbs blocked platelet aggregation induced by adenosine diphosphate (ADP) (P > .5). Conversely, anti-GPIIb-IIIa MoAb LJ-CP8 inhibited platelet aggregation induced by ADP (IC50 1 mumol/L, but failed to block ristocetin-induced platelet aggregation (P > .5). In vivo, the intravenous infusion of anti-vWF MoAb BB3 BD5 or anti-GPIIb- IIIa MoAb LJ-CP8 into baboons at doses that abolished corresponding agonist-induced aggregation ex vivo (bolus injections of 0.5 mg/kg and 10 mg/kg, respectively) prolonged template bleeding times from baseline values of 4.0 +/- 0.3 minutes to > 27 +/- 4 minutes, and to > 26 +/- 4 minutes, respectively (P < .001 in both cases), without affecting the peripheral platelet count (P > .5). However, injection of anti-GPIb MoAb AP1 [10 mg/kg as IgG or 1 mg/kg as F(ab)2 fragments] produced immediate irreversible thrombocytopenia (< 40,000 platelets/microL). Anti-GPIIb-IIIa MoAb LJ-CP8 abolished platelet deposition and fibrin accumulation on collagen segments under both arterial and venous flow conditions (P < .01 in all cases), whereas MoAb BB3 BD5 produced minimal inhibition of platelet deposition and no decrease in fibrin accumulation at arterial shear rates and undetectable antithrombotic outcomes at low shear. Thus, inhibiting GPIIb-IIIa-dependent platelet recruitment abrogates both thrombus formation and platelet hemostatic function at both venous and arterial shear rates. By contrast, interfering with GPIb-vWF-dependent platelet interactions abolishes platelet hemostatic function without producing corresponding antithrombotic effects.     

Adhesion of platelets to surface-bound fibrinogen under flow

After platelet activation, fibrinogen mediates platelet-platelet interactions leading to platelet aggregation. In addition, fibrinogen can also function as a cell adhesion molecule, providing a substratum for adhesion of platelets and endothelial cells. In this report, we studied the adhesion of platelets to surface-immobilized fibrinogen under flow in different shear rates. Heparinized whole blood containing mepacrine-labeled platelets was perfused for two minutes at various wall shear rates from 250 to 2,000 s-1 in a parallel plate flow chamber. The number of adherent fluorescent platelets was quantitated every 15 seconds with an epifluorescent videomicroscope and digital image processing system. When compared with platelet adhesion and aggregation seen on glass surfaces coated with type I bovine collagen, a significant increase in platelet adhesion was observed on immobilized fibrinogen up to wall shear rates of 800 s-1. The adherent platelets formed a single layer on fibrinogen-coated surfaces. Under identical conditions, no significant adhesion was observed on fibronectin- or vitronectin-coated surfaces. Although platelet adhesion to collagen was substantially inhibited by the platelet inhibitors prostaglandin E1 and theophylline, these inhibitors had no effect on platelet adhesion to fibrinogen. Platelets adhered to recombinant homodimeric wild-type (gamma 400-411) fibrinogen, but not to the recombinant homodimeric gamma' variant of fibrinogen. Platelet adhesion to recombinant fibrinogen with RGD to RGE mutations at positions alpha 95-97 and alpha 572-574 was similar to that with plasma-derived fibrinogen. These results show that platelets adhere to fibrinogen-coated surfaces under moderate wall shear rates, that the interaction is mediated by the fibrinogen 400-411 sequence at the carboxy-terminus of the gamma chain, and that the interaction is independent of platelet activation and the RGD sequences in the alpha chain.     

Glycoprotein IIb-IIIa and RGD(S) are not important for fibronectin- dependent platelet adhesion under flow conditions

Previous studies have indicated that activated blood platelets interact with fibronectin through binding of fibronectin to the glycoprotein IIb- IIIa complex (GPIIb-IIIa). The cell attachment site of fibronectin with its crucial arg-gly-asp(-ser) [RGD(S)]sequence is involved in these bindings. We studied the importance of these interactions for the fibronectin dependence of platelet adhesion under flow conditions. An RGDS-containing hexapeptide (GRGDSP) was compared with a nonreactive control peptide (GRGESP). The GRGDSP-peptide inhibited thrombin-induced aggregation and adhesion under static conditions at 0.1 mmol/L. This concentration had no effect on platelet adhesion to nonfibrillar collagen type I in flow. GRGDSP at 1 mmol/L had a significant inhibitory effect at 1,500 s-1, but not at the lower shear rates of 800 and 300 s-1 where platelet adhesion is also fibronectin dependent. On the matrix of cultured human umbilical vein endothelial cells, 1 mmol/L GRGDSP had no effect on platelet adhesion. The relation between GPIIb- IIIa and fibronectin dependence was investigated with platelets of a patient with Glanzmann's thrombasthenia and monoclonal antibodies to GPIIb-IIIa using endothelial cell matrix (ECM) as a surface. Platelets of normal controls or a patient with Glanzmann's thrombasthenia showed a similar inhibition of adhesion in the presence of fibronectin-free plasma after the ECMs had been preincubated with antifibronectin F(ab')2 fragments. Incubation of platelets with anti-GPIIb-IIIa showed inhibition of platelet adhesion at high shear rates. Dependence on fibronectin for platelet adhesion was still observed even though separate experiments had shown that these anti-GPIIb-IIIa antibodies could block binding of radiolabeled fibronectin to thrombin-activated platelets. These data suggest the existence of another binding system for the interaction of platelets with fibronectin that may only appear when fibronectin is present on a surface.     

Shear-dependent functions of the interaction between soluble von Willebrand factor and platelet glycoprotein Ib in mural thrombus formation on a collagen surface

Recent flow studies have clearly established the function of von Willebrand factor (vWF) in initial platelet adhesion, in which the interaction of surface-immobilized vWF with platelet glycoprotein (GP) Ib, particularly at high shear rates, leads to the transient capture of flowing platelets onto the surface. This interaction is thought to trigger activation of platelet GP IIb/IIIa, leading to irreversible platelet adhesion and subsequent mural thrombus growth. The role of vWF-GP Ib interaction in secondary thrombus growth remains to be clarified, however. In this study, time-course images of the thrombus formation process were obtained using a whole blood flow system that allows real-time visualization of fluorescence-labeled platelet thrombus formation. The system employs a collagen-coated surface in a parallel plate flow chamber mounted on an epifluorescence microscope, which is then subjected to computer-assisted image analysis. In perfusion of blood preincubated with anti-vWF antibody NMC-4, which blocks the vWF-GP Ib interaction in solution, neither primary platelet adhesion nor subsequent thrombus growth on a collagen surface was detected at high shear rates (> or = 1210/s). In addition, even under experimental conditions in which initial platelet adhesion normally occurred, NMC-4-treated blood perfusion showed an apparent defect of secondary thrombus growth at the same high shear rates. The overall process of thrombus formation at low shear rates (< or = 340/s) was not affected by specific blockers of the vWF-GP Ib interaction. These findings indicate that, in addition to the interaction of surface-immobilized vWF with GP Ib in platelet adhesion, the interaction of soluble vWF with GP Ib is required for secondary thrombus growth selectively at high shear rates.     

Hybridoma antibodies to human von Willebrand factor

Hybridoma antibodies specific for seven independent topographical sites were used to characterize von Willebrand factor (vWF) and to relate the epitopes to functional loci required for vWF-mediated adhesion of platelets to subendothelium and ristocetin-induced platelet aggregation. The capacity of antibodies to influence the adhesion of human platelets to rabbit aortic subendothelium was analysed in annular perfusion chambers. At a high shear rate similar to that of the microcirculation, four monoclonal antibodies inhibited adhesion. In contrast, no inhibition was observed at low shear. Only one of the four antibodies that inhibited platelet adhesion also attenuated ristocetin-cofactor activity (VIIIR:RCo). Conversely, one antibody that inhibited VIIIR:RCo had no effect upon platelet adhesion. These data support the hypothesis that the molecular loci involved in the two biological functions of vWF are not identical. When these conclusions are considered within the context of a spatial map of the vWF protein surface developed by competitive displacement analysis, the epitopes related to platelet adhesion appear to be spaced and differ from those involved in ristocetin-induced platelet-platelet interaction.     

Platelet adhesion to fibronectin in flow: the importance of von Willebrand factor and glycoprotein Ib

We describe glycoprotein (GP) Ib as a mediator of adhesion to fibronectin, specifically in flow. A monoclonal antibody (MoAb) directed to the von Willebrand factor (vWF)-binding site on this receptor or the absence of this receptor on the platelet membrane, in the case of a patient with the Bernard-Soulier syndrome, reduced platelet coverage to fibronectin to approximately 30% of the control value. A MoAb directed to the GP Ib-binding site on vWF showed a similar effect. With washed platelets in the absence of plasma vWF, the inhibitory effect of the anti-GP Ib antibody was the same as with whole blood. No inhibition with the anti-GP Ib antibody was observed when we used blood from patients with severe von Willebrand disease (vWD) or from a patient with vWD type I (platelet low). Addition of vWF to vWD blood resulted in restoration of adhesion. Immunoelectron microscopy on platelets adhering to fibronectin showed that GP Ib was homogeneously distributed over the entire surface of the platelet. vWF was present at the central zone and the edges of the platelet and at the basal interface between the platelet and the fibronectin surface. No direct binding of vWF to fibronectin could be demonstrated. These data indicate that GP Ib-mediated adhesion to fibronectin fully depends on vWF and that normal levels of plasma or platelet vWF are sufficient for optimal adhesion to fibronectin. The data suggest that the presence of platelets during perfusion is a prerequisite for vWF to support platelet adhesion to fibronectin.     

Deficiency of platelet membrane glycoprotein Ia associated with a decreased platelet adhesion to subendothelium: a defect in platelet spreading

A bleeding disorder with absent collagen-induced platelet aggregation and adhesion has been described in a patient whose platelets failed to express surface glycoprotein Ia. We studied the interaction of her platelets with subendothelium in an annular perfusion chamber and the interaction with purified human collagen type III in a rectangular perfusion system under flow conditions. Platelet adherence was almost completely absent both at low and high shear rates. The few platelets which adhered remained in the contact stage without subsequent spreading and aggregate formation. Addition of a monoclonal antibody, which was directed against the von Willebrand moiety of FVIII-VWF, to the blood, completely abolished platelet adherence at high shear rates and had a partial effect at low shear rates. These data indicate that von Willebrand factor plays a role in the initial attachment (contact stage) of platelets to subendothelium. We conclude that the bleeding disorder and excessively prolonged bleeding time in our patient are caused by a new specific defect of the platelet-vessel wall interaction.     

Role of von Willebrand factor associated to extracellular matrices in platelet adhesion

The respective role of plasmatic and endothelial extracellular matrix (ECM)-associated von Willebrand factor (vWF) in platelet adhesion was investigated at a high shear rate using a parallel-plate perfusion chamber. Incubation of the endothelial ECM with a monoclonal antibody (MoAb) to vWF, which specifically blocks vWF binding to platelet GP Ib (MoAb 322), inhibited 45% of platelet adhesion. Complete inhibition was achieved by incubating both plasma and endothelial ECM with MoAb 322 at concentrations that blocked only about 50% of adhesion when added separately. The effect of ECM-associated vWF was further demonstrated when a fibroblastic ECM, normally devoid of vWF, was coated with purified plasmatic vWF. Matrix associated-vWF was able to significantly enhance platelet adhesion in both the presence and the absence of plasmatic vWF. In contrast, this effect was not seen on endothelial ECM. Binding of exogenous vWF to the ECM was specific and dose dependent, reached the same value (500 ng/cm2) on both fibroblastic ECM and endothelial ECM, but exhibited a threefold-lower apparent dissociation constant (KD) on fibroblastic than on endothelial ECM. Our studies suggest that vWF deposited by endothelial cells in the ECM may be the most active form in platelet adhesion, whereas plasmatic vWF may only play a secondary role.     

Comparative in vitro efficacy of different platelet glycoprotein IIb/IIIa antagonists on platelet-mediated clot strength induced by tissue factor with use of thromboelastography: differentiation among glycoprotein IIb/IIIa antagonists

In the present study, the in vitro efficacy of different platelet glycoprotein IIb/IIIa (GPIIb/IIIa) antagonists on platelet-fibrin-mediated clot strength under shear was compared with the antiaggregatory efficacy by using tissue factor (TF) thromboelastography (TEG). The ability of platelets to augment the elastic properties of blood clots under shear conditions was measured by computerized TEG under conditions of maximal platelet activation accelerated by recombinant TF. Under these conditions, platelets significantly enhance clot strength 8-fold (relative to platelet-free fibrin clots). This effect was inhibited to a different extent by various platelet GPIIb/IIIa receptor antagonists; this inhibition appears to be dependent on the transmission of platelet contractile force to fibrin via the GPIIb/IIIa receptors. The GPIIb/IIIa antagonists with high binding affinity for resting and activated platelets and slow platelet dissociation rates (class I) but not those with fast platelet dissociation rates (class II) demonstrated potent and comparable inhibition of platelet aggregation and TF-TEG clot strength. Platelet GPIIb/IIIa antagonists of class I, such as XV459 (free-acid form of roxifiban), DMP802, XV454, and c7E3, demonstrated comparable inhibitory dose responses of TF-TEG clot strength and platelet aggregation, with an IC(50) of 50 to 70 nmol/L. In contrast, platelet GPIIb/IIIa antagonists from class II, with comparable antiaggregatory efficacy, such as DMP728, YZ202 (free-acid form of orbofiban), YZ211 (free-acid form of sibrafiban), YZ751, and other antagonists, have a much lower efficacy in altering the strength of TF-mediated clot formation (IC(50) >1.0 micromol/L). These data suggest differential efficacy among different GPIIb/IIIa antagonists in inhibiting platelet-fibrin clot retraction despite of equivalent antiaggregatory potency.     

Shear-induced platelet aggregation requires von Willebrand factor and platelet membrane glycoproteins Ib and IIb-IIIa

Different types of platelets in various types of plasma were subjected to levels of shear stress that produce irreversible platelet aggregation in normal platelet-rich plasma (PRP). At shear stresses of 90 or 180 dyne/cm2 applied for 30 seconds or five minutes, aggregation was either absent or only transient and reversible using severe von Willebrand's disease (vWD) PRP (less than 1% von Willebrand factor, vWF); Bernard-Soulier syndrome (BSS) PRP (platelets deficient in the membrane glycoprotein Ib, GPIb); normal PRP plus monoclonal antibody (MoAb) to GPIb; thrombasthenic PRP (platelets deficient in membrane glycoprotein IIb-IIIa complex, GPIIb-IIIa); and normal PRP plus MoAb to GPIIb-IIIa. Shear-induced aggregation was inhibited under the above conditions, even though the platelets were activated to release their granular contents. Sheared normal platelets in vWD plasma aggregated in response to added vWF. These studies demonstrate that the formation of stable platelet aggregates under conditions of high shear requires vWF and the availability of both GPIb and GPIIb-IIIa on platelet membranes. The experiments demonstrate that vWF-platelet interactions can occur in the absence of artificial agonists or chemical modification of vWF. They suggest a possible mechanism for platelet aggregation in stenosed or partially obstructed arterial vessels in which the platelets are subjected to relatively high levels of shear stress.