We have analyzed the multimeric structure of factor VIII/von Willebrand factor in plasma by sodium dodecyl sulfate electrophoresis using gels of varying porosity and a discontinuous buffer system. Factor VIII/von Willebrand factor bands were identified by reaction with 125I-labeled affinity-purified antibody and subsequent autoradiography. In 1% agarose gels, normal plasma displayed a series of sharply defined oligomers. However, increasing the agarose concentration to 2.0% or utilizing mixtures of 0.8% agarose--1.75% acrylamide revealed two bands of lesser intensity interposed between the major bands. When the acrylamide concentration in the gels was increased to 2.5%, bands with a faster mobility than IgM and fibronectin were now evident. Type IIA von Willebrand's disease showed not only an absence of the larger multimers but also a relative increase in several of the newly identified bands as compared to type IIB, type I, and normal. These studies suggest that factor VII/von Willebrand factor in IIA von Willebrand's disease is structurally different from that in other forms of the disorder. They also indicate that the multimeric composition of factor VII/von Willebrand factor is more complex than can be explained by simple linear polymerization of a single protomer.

A perspective on von Willebrand factor (vWF) 1 within a series on cell adhesion in vascular biology offers the opportunity to review the current understanding of platelet function in hemostasis and thrombosis. Platelets contribute to maintaining the normal circulation of blood through the preservation of vascular integrity and the control of hemorrhage after injury. Thus, the formation of platelet thrombi is a needed defense mechanism, but may precipitate diseases such as myocardial infarction in the setting of atherosclerosis (1, 2). Acute thrombotic arterial occlusion is the leading cause of morbidity and mortality in industrial societies, underscoring the relevance of studies aimed at unraveling how platelets respond to vascular injury. Particularly important in this regard is vWF, along with the subendothelial matrix components and membrane receptors that interact with it, owing to a key role in supporting unique aspects of platelet function. Indeed, vWF-dependent adhesion mechanisms can be viewed as the evolutionary adaptation to the need of establishing firm contact between a circulating element and the vessel wall meeting any mechanical challenge created by blood flow conditions. Platelet function: A paradigm of adhesion mechanisms for circulating vascular cellsPlatelets survey the inner lining of the vessel wall without interacting with it under normal circumstances, but respond rapidly to alterations of endothelial cells by attaching firmly to the site of lesion, where exposure of subendothelial structures may have occurred. A first layer of platelets adheres to the reactive surface, subsequently growing by accrual of additional platelets through homotypic aggregation. Both processes depend on the binding of membrane receptors to immobilized or soluble ligands, and are modulated by stimulus-coupled biochemical and cytoskeletal responses. Platelet thrombus formation is the paradigm of adhesion for circulating vascular cells, that can interact efficiently with the vessel wall only by withstanding opposing forces created by blood flow. In fact, the tendency of cells to move with the layer of fluid adjacent to the reactive surface creates unique biomechanical requirements for the formation of adhesive bonds. In a vessel, the velocity of blood near the wall is lower than towards the center, a difference resulting in a shearing effect between contiguous layers of fluid moving at different speed. Thus, "shear" is the consequence of the relative parallel motion of fluid planes during flow; it is greatest near the wall and decreases progressively towards the center of the vessel (3). The local shear rate is expressed in cm/s per cm, or the equivalent inverse second (s Ϫ 1 ). Fluid shear stress is force per unit area, the underlying cause of the shearing motion of blood. Shear rate is directly proportional to shear stress and inversely proportional to fluid viscosity. Assuming for blood a viscosity of 4 centipoise, the numerical value of shear stress (in dyn/cm 2 ) corresponds to shear rate divided by 40.The ro...

Background-Excessive bleeding may complicate congenital cardiac defects. To explain the pathogenesis of this abnormality, we evaluated selected parameters of primary hemostasis in patients with aortic valve stenosis before and after corrective surgery. Methods and Results-We examined shear-induced platelet aggregation with the filter aggregometer test and von Willebrand factor (vWF) structure by evaluating the multimeric distribution and extent of subunit proteolysis. The platelet count was reduced before corrective surgery, and shear-induced platelet aggregation was impaired. Moreover, vWF multimers of higher molecular mass were decreased, and proteolytic subunit fragments were increased. After correction of the cardiac defect, all of these parameters returned to normal. Conclusions-Alterations of vWF and platelet function may contribute to the bleeding diathesis in patients with aortic valve stenosis. Improvement after corrective surgery suggests that the passage of blood through a stenosed aortic valve may result in shear forces that induce vWF interaction with platelets in the circulation and, in turn, trigger platelet clearance, vWF degradation, and the impairment of primary hemostasis.

Twelve of 19 patients with myeloproliferative disorders showed a decrease of absence of the largest multimers of plasma von Willebrand factor (vWF) that correlated with elevated platelet counts but not with leukocyte counts. This suggested that platelets, rather than leukocytes, may be associated with the pathogenesis of the acquired vWF abnormality seen in these patients. To examine the hypothesis further, we studied 12 patients with reactive thrombocytosis after splenectomy. Increased platelet count (> 5 x 10(11)/L) after splenectomy was associated with vWF abnormalities indistinguishable from those detected in patients with myeloproliferative disorders. Accordingly, there was an inverse correlation between proportion of large vWF multimers and platelet, but not leukocyte, number: normalization of the platelet count was accompanied by restoration of a normal vWF multimeric pattern. These findings suggest that an increase in the number of platelets circulating in blood may favor the adsorption of larger vWF multimers onto the platelet membrane, resulting in their removal from the circulation and subsequent degradation.

An acquired hemorrhagic disorder developed in two patients in association with postsplenectomy thrombocytosis and leukocytosis during the course of the myeloproliferative syndrome. The presence of acquired von Willebrand's disease in these individuals was demonstrated by a decrease or absence of the larger von Willebrand factor (vWF) multimers, alteration of the repeating vWF multimeric “triplet,” decreased ristocetin cofactor activity (vWF:RCo), and prolonged bleeding time. The bleeding stopped in both patients after treatment with either 1-deamino-[8-D-arginine]-vasopressin (DDAVP) or Cohn fraction I. Treatment with thrombocytapheresis and azathioprine or busulfan resulted in reduction of the elevated platelet and white cell counts and was associated with partial correction of the vWF abnormalities and remission of the hemostatic abnormalities. In five additional patients with the myeloproliferative syndrome, but without bleeding symptoms, large multimers of plasma vWF were diminished also. These findings suggest that acquired von Willebrand's disease should be considered when a bleeding diathesis develops during the course of the myeloproliferative syndrome.

We have studied the modifications in the multimeric composition of plasma factor VIII/von Willebrand factor and the bleeding time response following administration of 1-Deamino-[8-D-arginine]-Vasopressin (DDAVP) to patients with different subtypes of von Willebrand's disease. In type I, all multimers were present in plasma in the resting state, though they were decreased in concentration. Administration of DDAVP resulted in an increased concentration of these forms as well as the appearance of larger forms than were previously present. There was concomitant correction of the bleeding time. In type IIA, large multimers were absent in the resting state, and although DDAVP induced an average threefold increase in the plasma concentration of factor VIII/von Willebrand factor, the larger multimers did not appear and the bleeding time, although shortened, was not corrected. In contrast, the larger multimers that were also absent from type IIB plasma in the resting state rapidly appeared following DDAVP administration. However, their appearance was transitory and the bleeding time, as in IIA patients, was shortened but not corrected. The characteristic multimeric composition of platelet factor VIII/von Willebrand factor in given subtypes predicted the alteration in plasma factor VIII/von Willebrand factor induced by DDAVP. These studies provide evidence that the different subtypes of von Willebrand's disease represent distinct abnormalities of factor VIII/von Willebrand factor. They also suggest that complete hemostatic correction following DDAVP can be routinely expected only in type I von Willebrand's disease, and only if factor VIII/von Willebrand factor can be raised to normal levels.

We have studied three afibrinogenemic patients, who had only trace amounts of plasma and platelet fibrinogen as measured by radioimmunoassay, and demonstrate here that the residual aggregation observed in their platelet-rich plasma is dependent upon von Willebrand factor (vWF) binding to the platelet membrane glycoprotein (GP)IIb/IIIa complex. The abnormality of aggregation was more pronounced when ADP, rather than thrombin, collagen, or the combination of ADP plus adrenaline was used to stimulate platelets. With all stimuli, nevertheless, the platelet response was completely inhibited by a monoclonal antibody (LJP5) that is known to block vWF, but not fibrinogen binding to GPIIb/IIIa. Addition of purified vWF to the afibrinogenemic plasma resulted in marked increase in the rate and extent ofaggregation, particularly when platelets were stimulated with ADP. This response was also completely blocked by LJP5.Addition of fibrinogen, however, restored normal aggregation even in the presence of IPS, a finding consistent with the knowledge that antibody LJP5 has no effect on platelet aggregation mediated by fibrinogen binding to GPIIb/IIIa. Two patients gave their informed consent to receiving infusion of 1-desamino--D-arginine vasopressin (DDAVP), a vasopressin analogue known to raise the vWF levels in plasma by two-to fourfold. The bleeding time, measured before and 45 min after infusion, shortened from >24 min to 12 min and 50 s in one patient and from 16 min to 9 min and 30 s in the other. Concurrently, the rate and extent of ADP-induced platelet aggregation improved after DDAVP infusion. The pattern, however, reversed to baseline levels within 4 h. The concentration of plasma vWF increased after DDAVP infusion, but that of fibrinogen remained at trace levels. We conclude that vWF interaction with GPIIb/Il~a mediates platelet-platelet interaction and may play a role in primary hemostasis.

Two likely mechanisms for the initiation of arterial platelet thrombus formation under conditions of elevated fluid shear stresses are: (1) excessive adhesion and aggregation of platelets from rapidly flowing blood onto the exposed sub-endothelium of injured, atherosclerotic arteries; or (2) direct, fluid shear stress-induced aggregation of platelets in constricted arteries with intact endothelial cells. Mechanism (1) was simulated using a parallel plate flow chamber, fibrillar collagen type I-coated slides, and mepacrine-labeled (fluorescent) platelets in whole blood anticoagulated with citrate, hirudin, unfractionated porcine heparin, or low molecular weight heparin flowing for 1 to 2 minutes at wall shear rates of 100 to 3,000 seconds-1 (4 to 120 dynes/cm2). The precise sequence of interactions among von Willebrand factor (vWF), glycoprotein (GP)Ib, and GPIIb-IIIa during platelet adhesion and subsequent aggregation were resolved by direct real-time observation using a computerized epifluorescence video microscopy system. Adhesion at high shear rates was the result of the adsorption of large vWF multimers onto collagen and the binding of platelet GPIb to the insolubilized vWF. Aggregation occurred subsequently and required the binding of ligands, including vWF via its RGD binding domain, to GPIIb-IIIa. Mechanism (2) was modeled by producing shear stresses of 90 to 180 dynes/cm2 in a rotational cone and plate viscometer, which aggregates platelets from platelet-rich- plasma (PRP) anti-coagulated with citrate, hirudin, or either type of heparin in reactions that require large vWF multimers, Ca2+, adenosine diphosphate, and both GPIb and GPIIb-IIIa. Both vWF-mediated shear- aggregation in PRP and platelet-collagen adhesion in flowing whole blood (anticoagulated with citrate and hirudin) are inhibited by two potentially useful anti-arterial thrombotic agents: polymeric aurin tricarboxylic acid (ATA; 28.5 to 114 micrograms/mL), which binds to vWF and inhibits its attachment of GPIb, and a recombinant vWF fragment (rvWF445–733; 30 to 200 micrograms/mL) that binds to platelet GPIb (in the absence of any modulator) and blocks attachment of vWF multimers. Unfractionated heparin, but not low molecular weight heparin, apparently binds to rvWF445–733 and counteracts the inhibitory effects of the vWF fragment in vitro on shear-aggregation and platelet-collagen adhesion.