Shilun Zheng

Analyses of cellular multimerin 1 receptors: in vitro evidence of binding mediated by αIibβ3 and αvβ3

Multimerin 1 (MMRN1) is a large, soluble, polymeric, factor V binding protein and member of the EMILIN protein family. In vivo, MMRN1 is found in platelets, megakaryocytes, endothelium and extracellular matrix fibers, but not in plasma. To address the mechanism of MMRN1 binding to activated platelets and endothelial cells, we investigated the identity of the major MMRN1 receptors on these cells using wild-type and RGE-forms of recombinant MMRN1.Ligand capture,cell adhesion,ELISA and flow cytometry analyses of platelet-MMRN1 binding, indicated that MMRN1 binds to integrins αIibβ 3 and αvβ3. Endothelial cell binding to MMRN1 was predominantly mediated by αvβ3 and did not require the MMRN1 RGD site or cellular activation.Like many other αvβ3 ligands, MMRN1 had the ability to support adhesion of additional cell types, including stimulated neutrophils. Expression studies, using a cell line capable of endothelial-like MMRN1 processing, indicated that MMRN1 adhesion to cellular receptors enhanced its extracellular matrix fiber assembly. These studies implicate integrin-mediated binding in MMRN1 attachment to cells and indicate that MMRN1 is a ligand for aIibβ 3 and αvβ3.

Fibrinogen degradation products in patients with the Quebec platelet disorder

The Quebec platelet disorder is a serious bleeding disorder associated with proteolysis of α‐granular proteins, including fibrinogen. We evaluated fibrinogen degradation product (FDP) assays as screening tests for this disorder. Patients with the Quebec platelet disorder (13/13) had elevated serum FDPs (P <0.01) due to secreted degraded platelet fibrinogen, but normal plasma FDPs and D‐dimers. Unrelated controls with bleeding disorders (32/34) had undetectable FDPs, and controls with FDPs due to disseminated intravascular coagulation (DIC) and other illnesses (11/11) had elevated FDPs in all assays (P < 0.01). Immunoblot analyses indicated plasma fibrinogen was normal in the Quebec patients and platelet FDPs were found in their platelet lysates, releasates and serum samples. Their platelet FDPs were not altered by treatment with fibrinolytic inhibitors and were different from the FDPs in DIC and in plasmin‐digested fibrinogen. Tests for serum FDPs may provide a simple rapid way to screen for the Quebec platelet disorder.

The storage defects in grey platelet syndrome and αδ‐storage pool deficiency affect α‐granule factor V and multimerin storage without altering their proteolytic processing

Among proteins stored in α‐granules, multimerin and factor V share unusual features: they bind to each other, are proteolysed to unique forms and are stored eccentrically in α‐granules. These unique features of their processing led us to study these proteins in alpha delta storage pool deficiency (αδ‐SPD) and grey platelet syndrome (GPS, α‐SPD), two conditions known to impair α‐granule protein storage. Platelet factor V and multimerin were severely reduced in GPS, whereas they ranged from reduced to normal in αδ‐SPD. The platelet levels of factor V and multimerin in these disorders indicated multimerin deficiency was not predictive of platelet factor V deficiency, although it reduced the amount of multimerin associated with platelet factor V. In GPS only, the defect in storing proteins was associated with increased multimerin and multimerin‐factor V complexes in plasma. Like normal platelets, GPS and αδ‐SPD platelets contained factor V mainly in granules. Platelet factor V and multimerin were proteolysed to normal platelet forms in GPS and αδ‐SPD platelets, indicating that these conditions preserve some aspects of normal α‐granule protein processing. Although we found factor V can be stored in platelets deficient in multimerin, our data indicate that multimerin storage influences the point at which multimerin binds factor V.

Human platelets contain forms of Factor V in disulfide-linkage with multimerin

Factor V is an essential cofactor for blood coagulation that circulates in platelets and plasma. Unlike plasma factor V, platelet factor V is stored complexed with the polymeric alpha-granule protein multimerin. In analyses of human platelet factor V on nonreduced denaturing multimer gels, we identified that approximately 25% was variable in size and migrated larger than single chain factor V, the largest form in plasma. Upon reduction, the unusually large, variably-sized forms of platelet factor V liberated components that comigrated with other forms of platelet factor V, indicating that they contained factor V in interchain disulfide-linkages. With thrombin cleavage, factor Va heavy and light chain domains, but not B-domains,were liberated from the components linked by interchain disulfide bonds, indicating that the single cysteine in the B-domain at position 1085 was the site of disulfide linkage. Since unusually large factor V had a variable size and included forms larger than factor V dimers, the data suggested disulfide-linkage with another platelet protein, possibly multimerin. Immunoprecipitation experiments confirmed that unusually large factor V was associated with multimerin and it remained associated in 0.5 M salt. Moreover, platelets contained a subpopulation of multimerin polymers that resisted dissociation from factor V by denaturing detergent and comigrated with unusually large platelet factor V, before and after thrombin cleavage. The disulfide-linked complexes of multimerin and factor V in platelets, which are cleaved by thrombin to liberate factor Va, could be important for modulating the function of platelet factor V and its delivery onto activated platelets. Factor Va generation and function from unusually large platelet factor V is only speculative at this time.