Platelet adhesion receptors and (patho)physiological thrombus formation

In thrombus formation associated with hemostasis or thrombotic disease, blood platelets first undergo a rapid transition from a circulating state to an adherent state, followed by activation and aggregation. Under flow conditions in the bloodstream, this process potentially involves platelet-platelet, platelet-endothelium, platelet-subendothelial matrix, and platelet-leukocyte interactions. Specific adhesion receptors on platelets mediate these interactions, by engaging counter-receptors on other cells, or noncellular ligands in the plasma or matrix. The glycoprotein (GP) Ib-IX-V complex on platelets initiates adhesion at high shear stress by binding the adhesive ligand, von Willebrand Factor (vWF). GP Ib-IX-V may also mediate platelet-endothelium or platelet-leukocyte adhesion, by recognition of P-selectin or Mac-1, respectively. Other membrane glycoproteins, such as the collagen receptor GP VI, may trigger platelet activation at low shear rates. Engagement of GP Ib-IX-V or GP VI leads ultimately to platelet aggregation mediated by the integrin, alphaIIbbeta3 (GP IIb-IIIa). This review will focus on recent advances in understanding structure-activity relationships of GP Ib-IX-V, its role in initiating thrombus formation, and its emerging relationships with other vascular cell adhesion receptors.     

Ability of Different Glycoprotein IIb-IIIa Ligands to Support Platelet Aggregation Induced by Activating Antibody CRC54

The ability of different ligands of glycoprotein (GP) IIb-IIIa (alphaIIb/beta3-integrin) to support platelet aggregation stimulated by activating anti-GP IIb-IIIa monoclonal antibody (monoAB) CRC54 has been investigated. Antibody CRC54 stimulated aggregation of washed platelets not only in the presence of fibrinogen, the main GP IIb-IIIa ligand, but also in the presence of von Willebrand factor (vWF). Unlike these ligands, fibronectin failed to support CRC54-induced aggregation. Fibrinogen and vWF dependent platelet aggregation was completely suppressed by GP IIb-IIIa antagonists--preparations Monafram (F(ab')2 fragments of monoAB that blocked GP IIb-IIIa receptor activity) and aggrastat (RGD-like peptidomimetic). However, aggregation stimulated in the presence of vWF was also completely inhibited by monoAB AK2 directed against GP Ib and capable of blocking its binding with vWF. CRC54-induced aggregation of platelets from patient with GP Ib deficiency in the presence of vWF was significantly lower than aggregation of platelets from normal donors and was not inhibited by anti-GP Ib antibody but still blocked by GP IIb-IIIa antagonist Monafram. Monafram also suppressed CRC54-stimulated platelet adhesion to plastic-adsorbed fibrinogen, vWF, and fibronectin. Unlike CRC54-induced platelet aggregation supported by fluid phase vWF, CRC54-induced adhesion to adsorbed vWF was not affected by anti-GP Ib antibody. Aggregation induced by CRC54 in the presence of fibrinogen and vWF was only partially suppressed by prostaglandin E1, an inhibitor of platelet activation, and was associated with serotonin release from platelet granules only when Ca2+ concentration was decreased from 1 mM (physiological level) to 0.1 mM. The data indicate that vWF supports CRC54-induced platelet aggregation via interaction with two receptors--GP IIb-IIIa and GP Ib. Aggregation induced by CRC54 in the presence of vWF or fibrinogen is only partially dependent on platelet activation and is accompanied with granule secretion only at low Ca2+ concentrations.     

Ristocetin-dependent reconstitution of binding of von Willebrand factor to purified human platelet membrane glycoprotein Ib-IX complex

Whether the human platelet membrane glycoprotein (GP) Ib-IX complex is the receptor for ristocetin-dependent binding of von Willebrand factor (vWF) has been examined by reconstitution with the purified components using a solid-phase bead assay. Purified GP Ib-IX complex was bound and orientated on the beads via a monoclonal antibody, FMC 25, directed against the membrane-associated region of the complex. Specific binding of 125I-labeled vWF to the GP Ib-IX complex coated beads was strictly ristocetin dependent with maximal binding occurring at ristocetin concentrations greater than or equal to 1 mg/mL. Ristocetin-dependent specific binding of 125I-labeled vWF was saturable. The observed binding was specific to the interaction between vWF and the GP Ib-IX complex since there was no ristocetin-dependent specific binding of vWF if the physicochemically related platelet membrane glycoprotein, GP IIb, was substituted for the GP Ib-IX complex in a corresponding bead assay. Further, neither bovine serum albumin nor other adhesive glycoproteins, such as fibrinogen or fibronectin, specifically bound to the GP Ib-IX complex in the presence of ristocetin. Ristocetin-dependent binding of vWF to platelets and to GP Ib-IX complex coated beads was inhibited by monoclonal antibodies against a 45,000 molecular weight N-terminal region of GP Ib but not by monoclonal antibodies directed against other regions of the GP Ib-IX complex. Similar correspondence between platelets and purified GP Ib-IX complex with respect to the ristocetin-dependent binding of vWF was obtained with anti-vWF monoclonal antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Signalling through the platelet glycoprotein Ib-V-IX complex

The glycoprotein Ib-V–IX is one of the major adhesive receptors expressed on the surface of circulating platelets. It is composed of four different polypeptides—GPIb, GPIbβ, GPIX, and GPV—and represents a multifunctional receptor able to interact with a number of ligands, including the adhesive protein von Willebrand factor, the coagulation factors thrombin, factors XI and XII, and the membrane glycoproteins P-selectin and Mac-1. Interaction of GPIb-V–IX with the subendothelial von Willebrand factor is essential for primary haemostasis, as it initiates platelet adhesion to the subendothelial matrix at the sites of vascular injury even under high flow conditions. Upon interaction with von Willebrand factor, GPIb-V–IX initiates transmembrane signalling events for platelet activation, which eventually result in integrin _IIbβ₃ stimulation and platelet aggregation. The investigation of the biochemical mechanisms for platelet activation by GPIb-V–IX has attracted increasing attention during the last years. This review will describe and discuss recent findings that have provided new insights into the events underlying GPIb-V–IX transmembrane signalling. In particular, it will summarise basic concepts on the structure of this receptor, extracellular ligands, and intracellular interactors potentially involved in transmembrane signalling. The recently suggested role of membrane Fc receptors in GPIb-V–IX-initiated platelet activation will also be discussed, along with the involvement of lipid metabolising enzymes, tyrosine kinases, and the cytoskeleton in the crosstalk between GPIb-V–IX and integrin _IIbβ₃.     

Glycoprotein Ib-IX-V

Glycoprotein (GP) Ib-IX-V is a remarkable platelet adhesion receptor of the leucine-rich repeat family. It has evolved to fulfil its major function of initiating platelet aggregation (thrombus formation) at high-shear stress in flowing blood. In addition to binding von Willebrand factor (vWF) in subendothelial matrix or plasma to trigger platelet aggregation, GPIb-IX-V also binds counter-receptors, alphaMbeta2 (Mac-1) on neutrophils or P-selectin on activated platelets or endothelial cells. GPIb-IX-V ligands also include alpha-thrombin, clotting factors XI/XIIa, and high-molecular-weight kininogen. Interactions involving GPIb-IX-V are therefore central to vascular processes of thrombosis and inflammation, and the receptor is under intense scrutiny as a potential therapeutic target.     

Suppressive Effect of CORM-2 on LPS-Induced Platelet Activation by Glycoprotein Mediated HS1 Phosphorylation Interference

In recent years, it has been discovered that septic patients display coagulation abnormalities. Platelets play a major role in the coagulation system. Studies have confirmed that carbon monoxide (CO) has important cytoprotective and anti-inflammatory function. However, whether CO could alter abnormal activation of platelets and coagulation and thereby reduce the incidence of mortality during sepsis has not been defined. In this report, we have used CO-releasing molecules (CORM-2) to determine whether CO inhibits LPS-induced abnormal activation of platelets and have explored the potential mechanisms. LPS was used to induce activation of platelets in vitro, which were purified from the peripheral venous blood of healthy adult donors. CORM-2 was applied as a potential therapeutic agent. CORM-2 preconditioning and delayed treatment were also studied. We found that in the LPS groups, the function of platelets such as spreading, aggregation, and release were enhanced abnormally. By contrast, the platelets in the CORM-2 group were gently activated. Further studies showed that the expression of platelet membrane glycoproteins increased in the LPS group. Coincidently, both hematopoietic lineage cell-specific protein 1 and its phosphorylated form also increased dramatically. These phenomena were less dramatically seen in the CORM-2 groups. Taken together, we conclude that during LPS stimulation, platelets were abnormally activated, and this functional state may be associated with the signal that is transmitted between membrane glycoproteins and HS1. CORM-released CO suppresses the abnormal activation of platelets by interfering with glycoprotein-mediated HS1 phosphorylation.     

Regulation of von Willebrand factor binding to the platelet glycoprotein Ib-IX by a membrane skeleton-dependent inside-out signal

The platelet receptor for von Willebrand factor (vWF), glycoprotein Ib-IX (GPIb-IX), mediates initial platelet adhesion and activation. We show here that the receptor function of GPIb-IX is regulated intracellularly via its link to the filamin-associated membrane skeleton. Deletion of the filamin binding site in GPIb(alpha) markedly enhances ristocetin- (or botrocetin)-induced vWF binding and allows GPIb-IX-expressing cells to adhere to immobilized vWF under both static and flow conditions. Cytochalasin D (CD) that depolymerizes actin also enhances vWF binding to wild type GPIb-IX. Thus, vWF binding to GPIb-IX is negatively regulated by the filamin-associated membrane skeleton. In contrast to native vWF, binding of the isolated recombinant vWF A1 domain to wild type and filamin binding-deficient mutants of GPIb-IX is comparable, suggesting that the membrane skeleton-associated GPIb-IX is in a state that prevents access to the A1 domain in macromolecular vWF. In platelets, there is a balance of membrane skeleton-associated and free forms of GPIb-IX. Treatment of platelets with CD increases the free form and enhances vWF binding. CD also reverses the inhibitory effects of prostaglandin E1 on vWF binding to GPIb-IX. Thus, GPIb-IX-dependent platelet adhesion is doubly controlled by vWF conformation and a membrane skeleton-dependent inside-out signal.     

Influence of cysteine proteinase inhibitors on platelet and plasma components of blood coagulation system

Factor XIIIa plays an important role in stabilization of formed fibrin clot during blood coagulation. Recent studies proved that factor XIIIa affects formation of coated platelets, which are highly procoagulant and characterized by a high level of alpha-granular proteins on their surface and expose surface phosphatidylserine after platelet activation. The ability of newly found cysteine proteinase inhibitors (CPIs) from plants to affect thiol group of the factor XIIIa active centre was recently discovered. Here, the effect of CPIs on the formation of coated platelets and activity of plasma components during blood coagulation process was investigated. It was found that CPIs dose-dependently decreased the fraction of coated platelets in the total platelet population during platelet activation and decreased endogenous thrombin potential (ETP) by 40% for thrombin generation in platelet-rich as well as in platelet-poor plasma. Such decrease of ETP could not be explained by the CPIs influence on factor XIIIa. Investigation of the effects of these inhibitors on factor Xa and thrombin activity has shown that CPIs dose-dependently inhibited their activity and might cause an ETP decrease. Thus, the obtained data indicated that CPIs affected both platelet and plasma components of blood coagulation system.     

Surface plasmon resonance detection of blood coagulation and platelet adhesion under venous and arterial shear conditions

A surface plasmon resonance (SPR) based flow chamber device was designed for real time detection of blood coagulation and platelet adhesion in platelet rich plasma (PRP) and whole blood. The system allowed the detection of surface interactions throughout the 6mm length of the flow chamber. After deposition of thromboplastin onto a section of the sensor surface near the inlet of the flow chamber, coagulation was detected downstream of this position corresponding to a SPR signal of 7 to 8 mRIU (7 to 8 ng/mm2). A nonmodified control surface induced coagulation 3.5 times slower. Platelet adhesion to gold and fibrinogen coated surfaces in the magnitude of 1.25 and 1.66 mRIU was also shown with platelets in buffer, respectively. SPR responses obtained with PRP and whole blood on surfaces that were methylated or coated with von Willebrand factor (vWF), fibrinogen, or collagen, coincided well with platelet adhesion as observed with fluorescence microscopy in parallel experiments. The present SPR detection equipped flow chamber system is a promising tool for studies on coagulation events and blood cell adhesion under physiological flow conditions, and allows monitoring of short-range surface processes in whole blood.     

Von Willlebrand Adhesion to Surfaces at High Shear Rates Is Controlled by Long-Lived Bonds

Von Willebrand factor (vWF) adsorbs and immobilizes platelets at sites of injury under high-shear-rate conditions. It has been recently demonstrated that single vWF molecules only adsorb significantly to collagen above a threshold shear, and here we explain such counterintuitive behavior using a coarse-grained simulation and a phenomenological theory. We find that shear-induced adsorption only occurs if the vWF-surface bonds are slip-resistant such that force-induced unbinding is suppressed, which occurs in many biological bonds (i.e., catch bonds). Our results quantitatively match experimental observations and may be important to understand the activation and mechanical regulation of vWF activity during blood clotting.     

Von Willlebrand Adhesion to Surfaces at High Shear Rates Is Controlled by?Long-Lived Bonds

Von Willebrand factor (vWF) adsorbs and immobilizes platelets at sites of injury under high-shear-rate conditions. It has been recently demonstrated that single vWF molecules only adsorb significantly to collagen above a threshold shear, and here we explain such counterintuitive behavior using a coarse-grained simulation and a phenomenological theory. We find that shear-induced adsorption only occurs if the vWF-surface bonds are slip-resistant such that force-induced unbinding is suppressed, which occurs in many biological bonds (i.e., catch bonds). Our results quantitatively match experimental observations and may be important to understand the activation and mechanical regulation of vWF activity during blood clotting.     

Identification of aspartic acid 514 through glutamic acid 542 as a glycoprotein Ib-IX complex receptor recognition sequence in von Willebrand factor. Mechanism of modulation of von Willebrand factor by ristocetin and botrocetin.

As the first step in hemostasis, the binding of von Willebrand factor (vWF) to the platelet membrane glycoprotein (GP) Ib-IX complex is essential for platelet adhesion at high-shear blood flow. This interaction in vivo requires the prior binding of vWF to the subendothelial matrix, a process which exposes a normally cryptic binding site on vWF for the GP Ib-IX complex. This process can be mimicked in vitro by modulators such as ristocetin or the snake venom protein botrocetin or by desialation of vWF. We have previously localized the GP Ib binding site on vWF to a monomeric dispase fragment which extends from Leu-480/Val-481 to Gly-718 in the primary sequence of mature vWF [Andrews, R. K., Gorman, J. J., Booth, W. J., Corino, G. L., Castaldi, P. A., & Berndt, M. C. (1989) Biochemistry 28, 8326-8336]. This fragment also contains a distinct binding site for botrocetin. Analysis of synthetic peptides corresponding to hydrophilic stretches of sequence within this fragment indicated that the sequence Asp-514-Glu-542 represents a major adhesive sequence involved in receptor recognition. This peptide inhibited both the ristocetin- and botrocetin-mediated binding of vWF to either platelets or purified GP Ib-IX complex (IC50 approximately 50-200 microM) as well as the asialo-vWF- and bovine vWF-dependent agglutination of platelets. Both the N- and C-terminal halves of the peptide were inhibitory but less so than the intact peptide. This peptide also inhibited botrocetin binding to vWF, suggesting that botrocetin modulates vWF-GP Ib interaction by binding in close proximity to the vWF adhesion sequence.(ABSTRACT TRUNCATED AT 250 WORDS)     

von Willebrand factor unfolding mediates platelet deposition in a model of high-shear thrombosis

Thrombosis under high-shear conditions is mediated by the mechanosensitive blood glycoprotein von Willebrand factor (vWF). vWF unfolds in response to strong flow gradients and facilitates rapid recruitment of platelets in flowing blood. While the thrombogenic effect of vWF is well recognized, its conformational response in complex flows has largely been omitted from numerical models of thrombosis. We recently presented a continuum model for the unfolding of vWF, where we represented vWF transport and its flow-induced conformational change using convection-diffusion-reaction equations. Here, we incorporate the vWF component into our multi-constituent model of thrombosis, where the local concentration of stretched vWF amplifies the deposition rate of free-flowing platelets and reduces the shear cleaning of deposited platelets. We validate the model using three benchmarks: in vitro model of atherothrombosis, a stagnation point flow, and the PFA-100, a clinical blood test commonly used for screening for von Willebrand disease (vWD). The simulations reproduced the key aspects of vWF-mediated thrombosis observed in these experiments, such as the thrombus location, thrombus growth dynamics, and the effect of blocking platelet-vWF interactions. The PFA-100 simulations closely matched the reported occlusion times for normal blood and several hemostatic deficiencies, namely, thrombocytopenia, vWD type 1, and vWD type 3. Overall, this multi-constituent model of thrombosis enables macro-scale 3D simulations of thrombus formation in complex geometries over a wide range of shear rates and accounts for qualitative and quantitative hemostatic deficiencies in patient blood.     

Human cytomegalovirus infection of endothelial cells triggers platelet adhesion and aggregation

Human cytomegalovirus (HCMV) is an opportunistic pathogen that has been implicated in the pathogenesis of vascular diseases. HCMV infection of endothelial cells may lead to vascular damage in vitro, and acute-phase HCMV infection has been associated with thrombosis. We hypothesized that viral infection of endothelial cells activates coagulation cascades and contributes to thrombus formation and acute vascular catastrophes in patients with atherosclerotic disease. To assess the effects of HCMV on thrombogenesis, we examined the adhesion and aggregation of blood platelets to uninfected and HCMV-infected endothelial cells. At 7 days after infection, platelet adherence and aggregation were greater in infected than in uninfected cultures (2,000 platelets/100 cells and 225 +/- 15 [mean +/- standard error of the mean] aggregates/five microscopic fields versus 100 platelets/100 cells and no aggregates). von Willebrand factor (vWF), ICAM-1, and VCAM-1 but not collagen IV, E-selectin, P-selectin, CD13, and CD31 were expressed at higher levels on infected cells than on uninfected cells. Platelet aggregation was inhibited by blocking of platelet GPIb (with blocking antibodies) or GPIIb/IIIa (with ReoPro) or by blocking of vWF (with polyclonal antibodies to vWF). Furthermore, blocking of vWF, platelet GPIb, and ICAM-1 but not of the endothelial cell marker CD13, alpha(5)beta(3)-integrin, or HCMV glycoprotein B reduced platelet adherence to infected cells by 75% +/- 5%, 74% +/- 5%, or 18% +/- 5%, respectively. The increased thrombogenicity was dependent on active virus replication and could be inhibited by foscarnet and ganciclovir; these results suggest that a late viral gene may be mediating this phenomenon, which may contribute to vascular catastrophes in patients with atherosclerotic disease.     

Cargo selectivity of the ERGIC-53/MCFD2 transport receptor complex

Exit of soluble secretory proteins from the endoplasmic reticulum (ER) can occur by receptor-mediated export as exemplified by blood coagulation factors V and VIII. Their efficient secretion requires the membrane lectin ER Golgi intermediate compartment protein-53 (ERGIC-53) and its soluble luminal interaction partner multiple coagulation factor deficiency protein 2 (MCFD2), which form a cargo receptor complex in the early secretory pathway. ERGIC-53 also interacts with the two lysosomal glycoproteins cathepsin Z and cathepsin C. Here, we tested the subunit interdependence and cargo selectivity of ERGIC-53 and MCFD2 by short interference RNA-based knockdown. In the absence of ERGIC-53, MCFD2 was secreted, whereas knocking down MCFD2 had no effect on the localization of ERGIC-53. Cargo binding properties of the ERGIC-53/MCFD2 complex were analyzed in vivo using yellow fluorescent protein fragment complementation. We found that MCFD2 is dispensable for the binding of cathepsin Z and cathepsin C to ERGIC-53. The results indicate that ERGIC-53 can bind cargo glycoproteins in an MCFD2-independent fashion and suggest that MCFD2 is a recruitment factor for blood coagulation factors V and VIII.     

Modulation of platelet function through adhesion receptors. A dual role for glycoprotein IIb-IIIa (integrin alpha IIb beta 3) mediated by fibrinogen and glycoprotein Ib-von Willebrand factor.

We have found that the form of glycoprotein (GP) IIb-IIIa (integrin alpha IIb beta 3) expressed on nonstimulated platelets is a functional receptor that mediates selective and irreversible adhesion to immobilized fibrinogen. This occurs even in the presence of the elevated intracellular cAMP levels induced by prostaglandin E1 or after inhibition of protein kinase C activity by sphingosine. In the absence of inhibitors, platelets adhering to fibrinogen through GP IIb-IIIa become fully activated and aggregate with one another. Immobilized von Willebrand factor (vWF), in contrast, is recognized by nonstimulated platelets through another receptor, GP Ib. This interaction leads to a change in the ligand recognition specificity of GP IIb-IIIa that can then bind to immobilized vWF and mediate irreversible platelet adhesion and aggregation; this process, however, is inhibited by elevated intracellular cAMP levels or blockade of protein kinase C activity. Therefore, GP Ib and GP IIb-IIIa induce platelet activation through the selective recognition of immobilized vWF and fibrinogen, respectively, in the absence of exogenous agonists. Moreover, "nonactivated" and "activated" GP IIb-IIIa exhibits distinctly different reactivity toward surface-bound vWF, and the functional switch can be induced by the binding of vWF to GP Ib. These findings demonstrate the modulation of platelet function by two different adhesion receptors, GP Ib and GP IIb-IIIa, as well as the distinct dual role of the latter as the necessary common mediator of irreversible adhesion and aggregation on both fibrinogen and vWF.     

Characterization of recombinant von Willebrand factor corresponding to mutations in type IIA and type IIB von Willebrand disease.

Type IIA and IIB von Willebrand disease (vWD) result from defects in von Willebrand factor (vWF). Although both type IIA and IIB vWD are characterized by the absence of high molecular weight multimers in plasma, vWF from patients with type IIA vWD demonstrates a decreased affinity for the platelet receptor glycoprotein Ib (GPIb), whereas vWF from patients with type IIB vWD show an increased affinity for GPIb. To investigate how structural alterations in vWF affect its interaction with GPIb, we reproduced the reported potential mutations in type IIA and IIB vWD in vWF cDNA and expressed the recombinant proteins in COS-1 cells. The type IIA vWF potential mutation was represented by a G-->A transversion which results in the substitution of Lys for Glu at position 875 in the mature vWF subunit (rvWFLys875). The type IIB vWF mutation corresponds to a duplicated ATG codon, resulting in three contiguous methionines starting at position 540-541 in the normal vWF sequence (rv-WFduplMet540-541). The subunit composition and multimeric structure of both mutant proteins were similar to the wild-type rvWF. The rvWFLys875 bound to fixed platelets in the presence of ristocetin similar to wild-type rvWF. The rvWFduplMet540-541 bound to fixed platelets in the absence of agonist. The rvWFLys875 appears to interact normally with GPIb, and the decreased affinity for the platelet receptor observed in plasma is probably a consequence of prior reduction in multimeric size resulting from the defect. In contrast, the duplication of Met540-541 increases the reactivity of vWF for its platelet receptor.     

Platelet activation as a marker for in vivo prothrombotic activity: detection by flow cytometry

Circulating platelets play a pivotal role in hemostasis. The platelet hemostatic function involves the direct interaction with damaged vessel walls, and circulating coagulation factors, primarily thrombin resulting in platelet activation, aggregation and formation of hemostatic plug. Flow cytometry is a useful technique for the study of platelet activation in circulating blood. Platelet activation markers for ex vivo analysis may include a) activation-dependent epitopes of the membrane glycoprotein (GP) IIb/IIIa (CD41a) receptor, as demonstrated by the binding of activation-specific monoclonal antibodies (MoAbs) PAC1, anti-LIBS1 and anti-RIBS); b) the expression of P-selectin (CD62p), the alpha-granule GP translocated to the platelet surface following release reaction; and c) platelet procoagulant activity, as demonstrated by the binding of i) annexin V protein to the prothrombinase-complex (prothrombin, activated factor X (Xa) and V (Va)) binding sites on the surface of activated platelets, and of ii) MoAbs against activated coagulation factors V and X bound to the surface of activated platelets. Using this method, platelet activation as a marker for in vivo prothrombotic activity can be demonstrated in various clinical conditions including coronary angioplasty, orthostatic challenge in primary depression, sickle cell disease in clinical remission and during pain episode, and in pregnancy-related hypertension with marked increase during preeclampsia. The finding of platelet procoagulant activity is corroborated by increased levels of plasma markers for thrombin generation and fibrinolytic activity.     

The role of von Willebrand factor multimers and propeptide cleavage in binding and stabilization of factor VIII

von Willebrand factor (vWF) is a multimeric glycoprotein that promotes platelet aggregation and stabilizes coagulation factor VIII in the plasma. vWF is also required for the stable accumulation of recombinant factor VIII secreted from cells in a heterologous expression system. In this report, we show that vWF can promote the in vitro reconstitution of factor VIII activity from dissociated heavy and light chains of factor VIII, suggesting that vWF may act to promote stable assembly of factor VIII subunits at the site of secretion. The structural requirements for vWF propeptide cleavage and for vWF multimerization in its binding and stabilization of factor VIII was examined using specifically altered recombinant vWF. The mutant vWF molecules were also assayed for their function in ristocetin-induced platelet agglutination mediated through the platelet receptor GPIb. Deletion of the vWF propeptide produced a dimeric vWF molecule that failed to mediate platelet agglutination, suggesting that multimerization is required for vWF to attain functional GPIb binding. This mature dimeric form of vWF, however, was fully capable of binding to and supporting stable secretion of factor VIII. A vWF mutant with an altered propeptide cleavage site formed large multimers of uncleaved pro-vWF that functioned in platelet agglutination. However, this noncleavage mutant neither bound to or supported stable accumulation of factor VIII. Analysis of the vWF propeptide, expressed independently, demonstrated that it could not bind factor VIII or stabilize its secretion. These results show that the dimeric mature vWF subunit is sufficient to bind and stabilize factor VIII and that the presence of uncleaved vWF propeptide inhibits both factor VIII binding and stabilization.