Sybil B. Williams

Platelet von Willebrand factor: Comparison with plasma von Willebrand factor

Platelet von Willebrand factor (vWf) was compared to its plasma counterpart. The platelet vWf was different from plasma vWf in that the multimeric organization was different, larger multimers were present, and the ratio of vWf activity to antigen was higher. Platelet and plasma vWf were similar in their antigenic reactivity in the electroimmunoassay and by liquid phase radioimmunoassay. The amount of vWf activity in large platelets was significantly higher than in normal platelets while the antigen content, although somewhat higher, was not significant. These studies show differences between normal platelet and plasma vWf, and also suggest that platelet size must be considered when platelet vWf is measured in disease states.

Purification and characterization of human platelet von Willebrand factor

Summary. Platelet von Willebrand factor (vWf) was purified from human platelet concentrates. The multimeric structure of the purified platelet vWf was similar to that observed in the initial platelet lysate, and, like the platelet lysate, the purified platelet vWf contained higher molecular weight multimers than plasma vWf. The apparent molecular weight of the reduced platelet vWf subunit was similar to the plasma vWf subunit. The N‐terminal amino acid of the purified platelet and plasma vWf was blocked. In concentration dependent binding to botrocetin‐ or ristocetin‐stimulated platelets, 125I‐plasma vWf bound with a higher affinity than platelet. The ristocetin cofactor activity per mg of purified plasma vWf was 5‐fold greater than the platelet vWf activity. Platelet and plasma vWf bound to collagen with similar affinities; however, platelet vWf bound to thrombin‐stimulated platelets and to heparin with a higher affinity than plasma vWf. The differences in the binding affinity(s) of plasma and platelet vWf to platelet GPIb and GPIIb/IIIa and extracellular matrix proteins may reflect different roles for plasma and platelet vWf in the initial stages of haemostasis and thrombosis.

Platelet von Willebrand Factor

von Willebrand factor (vWF) circulates in the blood in two distinct compartments. One, plasma vWF, is synthesized and released from endothelial cells; the second, synthesized by megakaryocytes, circulates in platelets primarily stored in the alpha granules. Recent experimental and clinical studies of von Willebrands disease (vWD) indicate that platelet vWF plays an important role in the bleeding time determination and the degree of clinical bleeding in vWD. Platelet vWF is released from the platelet alpha granules by various agonists and then rebinds to the glycoprotein IIb/IIIa complex. Fibrinogen or monoclonal antibodies against this complex inhibit 60 to 70% of the expression of platelet vWF. Aspirin inhibits 80% of the adenosine diphosphate-induced platelet vWF surface expression, and the platelet vWF surface expression that is not inhibited by aspirin can be almost totally inhibited by disruption of the platelet cytoskeleton. Platelet vWF may, in part, be expressed in the open canalicular system prebound to a receptor. Transfusion studies have shown that correction of the bleeding time in severe vWD requires both plasma and platelet vWF. On the basis of numerous studies, we hypothesize that platelet vWF plays an important role in platelet interaction with the subendothelial surfaces under conditions of high shear stress. After platelet contact, platelet vWF is released, binds to the glycoprotein IIb/IIIa complex, and forms a bridge between the subendothelial surface and the platelet, which initiates and supports platelet spreading. Platelet vWF also acts as an intercellular bridge between platelets and thereby promotes platelet aggregation. This process is important not only in the initial steps of hemostasis but also in the process of thrombosis.

Identification of a new mutation in platelet glycoprotein IX (GPIX) in a patient with Bernard–Soulier syndrome

We describe a new mutation in glycoprotein IX (GPIX) in a patient with Bernard–Soulier syndrome (BSS). Sequencing of GPIX revealed a homozygous (T→C) transition at nucleotide 1717 (GenBank/HUMGPIX/M80478), resulting in a Cys8 (TGT)→Arg (CGT) replacement in the mature peptide. DNA restriction enzyme analysis using BsaAI revealed that the patient was homozygous and that his parents were heterozygous for the defect. This mutation disrupts a putative disulphide bond between the Cys8 and Cys12 that would alter the secondary structure of GPIX and which probably accounts for the absence of the GPIb/IX/V complex from the platelet surface in this patient.

Platelet adhesion at high shear rates: The roles of von Willebrand factor/GPIb and the β1 integrin α2β1

We have previously described a monomeric rvWf fragment, Leu504-Lys728 that contains one disulfide bond linking Cys509-Cys695. This fragment, VCL, has previously been shown to inhibit vWf-ristocetin, asialo-vWf, and botrocetin-induced vWf binding and aggregation of platelets. VCL inhibited 50% of vWf binding to heparin, but it did not inhibit vWf binding to type I collagen. At a high shear force (2600-1 sec), VCL inhibited platelet adhesion to the subendothelial surface of human umbilical arteries. The maximum inhibition of platelet adhesion was 83 +/- 4% at a VCL concentration of 7.6 mumol/L. Various monoclonal anti-Very Late Activation antigens (VLA) antibodies were added to the VCL and tested for their ability to enhance the inhibition of platelet adhesion at high shear forces. Of all of the VLA antibodies tested, only the anti-VLA-2 antibody (176D7) inhibited platelet aggregation in the absence of VCL and enhanced the inhibition of platelet adhesion in the presence of VCL. The VLA-2 antibody and VCL together inhibited 96 +/- 4% of platelet adhesion at high shear forces.

Acquired hypoprothrombinemia: Effects of Danazol® treatment

The lupus anticoagulant may be accompanied by an acquired factor II deficiency and bleeding. We report on a patient with a lupus anticoagulant and factor II (FII) deficiency responsive to Danazol®. Acquired hypoprothrombinemia (FII) with the lupus anticoagulant (LA) may be accompanied by a hemorrhagic diathesis. A 64‐year‐old male with discoid lupus erythematosis bled after an intestinal polypectomy. His FII level was 18%, and his FII antigen level was 20%. Danazol® (D) (600 mg per day) administration was associated with a rise in FII activity and antigen to 50% within 10 days. The patient underwent abdominal surgery. We studied the effect(s) of D on the FII level and on other coagulation factors in this patient. The patients plasma FII antigen had a single precipitin arc compared to the two peaks of normal plasma on counterimmunoelectrophoresis with Ca++. The samples pre‐ and during D therapy had the same positively charged arc as normal samples, although they were quantitatively different. Neuraminidase treatment demonstrated a decrease in the positively charged migration of normal and the patients FII antigen. Affinity chromatography of normal and patient plasma on a Sepharose protein A column revealed FII antigen present in the patients bound fraction. The relative percentages of bound FII before and during D treatment were similar. During D therapy, levels of FIX and X rose 50‐100%, and protein C rose 20‐25%, while free protein S did not change. D is an effective therapy for acquired FII deficiency associated with LA. D does not affect the binding of Ig to FII, but D raises FII levels by increasing synthesis of the FII protein. (This article is a US Government work and, as such, is in the public domain in the United States of America.)

Inhibition of von willebrand factor binding to platelets by two recognition site peptides: the pentadecapeptide of the carboxy terminus of the fibrinogen gamma chain and the tetrapeptide arg-gly-asp-ser

When platelets are stimulated by thrombin or ADP, von Willebrand factor (vWf) binds to the platelet glycoprotein IIb/IIIa (GPIIb/IIIa). Two recognition sites for vWf binding have been identified on the GPIIb/IIIa. One is a tetrapeptide, arg-gly-asp-ser (RGDS), and the second is a dodecapeptide with the sequence HHLGGAKQAGDV. We used these purified peptides to analyze the recognition specificities for the platelet binding of vWf. The RGDS and the pentadecapeptide totally inhibited 125I-vWf in a dose-dependent manner. The IC50 was 8 microM for the RGDS and 40 microM for the pentadecapeptide. These results indicate that the RGDS binds with higher affinity to a vWf binding site on GPIIb/IIIa than does the pentadecapeptide site. When the two peptides were incubated together, they augmented the inhibition of vWf binding. These data support the evidence that the GPIIb/IIIa has two distinct binding sites for vWf binding. The RGDS site appears to have a higher affinity for the vWf binding site than does the pentadecapeptide site.

Platelet aggregation induced by type IIb platelet von Willebrand factor

Summary Platelet lysates from five patients with a form of type IIb von Willebrands disease (vWd), associated with spontaneous platelet aggregation and thrombocytopenia, induced platelet aggregation of normal and other vWds platelet‐rich plasma (PRP). Platelet lysate from normals, type I or type IIa vWd did not cause platelet aggregation of normal PRP. When polyclonal monospecific antibodies directed against plasma von Willebrand factor (vWf) were incubated with the type IIb platelet lysate, they inhibited the platelet aggregation. Monoclonal antibodies directed against the glycoprotein (GP) Ib binding domain of plasma vWf incubated with the type IIb platelet lysate did not inhibit the platelet aggregation. Normal platelets suspended in afibrinogenaemic plasma did not aggregate when type IIb vWd platelet lysate was added. Normal platelets incubated with monoclonal antibodies directed against the fibrinogen and vWf binding site(s) on the GPIIb/IIIa were not aggregated by the type IIb platelet lysate. Bernard‐Soulier PRP aggregated when type IIb vWd platelet lysate was added, while Glanzmanns thrombasthenic platelets did not. Peptides containing the RGDS sequence or the sequence of the carboxy terminal 15 amino acids of the gamma chain of fibrinogen inhibited the type IIb vWd platelet lysate‐induced platelet aggregation. These data suggest that type IIb platelet vWf can cause platelet aggregation of PRP without the addition of any agonist. This interaction is different from that observed with the plasma vWf from these patients.

Glycocalicin levels in the plasma of HIV+ patients : An indicator of platelet turnover

Glycocalicin (GC) is the carbohydrate-rich portion of platelet membrane glycoprotein Ib(alpha) that can be cleaved from circulating platelets by proteases. The plasma GC level is an indicator of platelet turnover. Using an ELISA for GC, we assayed the plasma of 20 normal children (age 6 to 13 years), 50 HIV+ children (ages 4 to 18 years), 32 normal adults (ages 21 to 53 years), and 50 HIV+ adults (ages 24 to 66 years). The results were adjusted for individual platelet counts to give GC indexes (GCI). The normal children and the normal adults had significantly different GCI distributions (P = .002). In both normal and HIV+ individuals the GCI decreased with increasing platelet count (-.73 < r < -.34). Twenty-eight percent of the HIV+ children and 28% of the HIV+ adults had elevated GCI values. The majority of these elevated values occurred in patients with platelet counts >100,000/microL. Neither the GCI nor the platelet count was correlated with viral load. The platelet count, however, was weakly correlated with the CD4 count in both children (r = .31) and adults (r = .30) infected with HIV. Also, the CD4 count was weakly and inversely correlated with GCI in HIV+ adults (r = -.34) and in children (r = -.24). We conclude that increased GCI and, by implication, increased platelet turnover is a relatively common feature of advanced HIV disease. Furthermore, GCI may be elevated in HIV+ patients even with a platelet count >100,000/microL, suggesting increased platelet turnover before thrombocytopenia develops.

Plasma Glycocalicin in Platelet Concentrates: Relationship to Other Parameters of the Storage Lesion

intact platelet membrane glycoprotein Ib (GPIb), of platelet concentrates over a period of 6 days and which is a critical protein for binding of von Willebrand factor (vWf). The reduction in GPIb integ- have analyzed the correlation of these parameters rity results from proteolytic cleavage of glycocalicin with the changes in GC. (GC), the carbohydrate-rich amino terminus of GPIb. GC has been shown to increase in the plasma of stored platelet concentrates (PCs) [2‐4]. 1. Materials and Methods