Farooq A. Siddiqui

The presence and release of tissue factor from human platelets

Tissue factor (TF) is a transmembrane receptor for FVII that triggers blood coagulation. It is not normally exposed to circulating blood, but may be produced by endothelium and monocytes under pathological conditions. Platelets take up TF-positive microparticles from leukocytes and TF appears on platelets adhering to leukocytes following collagen stimulation of blood. However, the presence of TF in circulating platelets has not been directly demonstrated. In this study, flow cytometric analysis of washed platelets from five healthy adult volunteers demonstrated TF-antigen on both resting platelets and platelets activated by thrombin (0.1 U/ml), collagen (5 w g/ml) or ADP (5 w M). TF released by platelets was demonstrated in the supernatants of non-activated and activated washed platelets by dot-immunoblotting and Western blotting. The amount of TF released from non-activated and activated platelets was quantitated using an enzyme-linked immunosorbent assay (ELISA). Washed non-activated and platelets activated by thrombin, collagen or ADP released 27-35 pg TF per mg protein. TF associated with the platelet surface was biologically inactive, although released TF was functionally active as determined by a two-stage factor X activation assay. We conclude that platelets contain an inactive form of TF that may develop functional activity following its release. However, the role of platelet TF in health and disease remains to be determined.

The role of CD40 in CD40L- and antibody-mediated platelet activation

Our initial finding that CD40- and CD40 ligand (CD40L)-deficient mice displayed prolonged tail bleeding and platelet function analyzer (PFA-100) closure times prompted us to further investigate the role of the CD40-CD40L dyad in primary hemostasis and platelet function. Recombinant human soluble CD40L (rhsCD40L), chemical cross-linking of which suggested a trimeric structure of the protein in solution, activated platelets in a CD40-dependent manner as evidenced by increased CD62P expression. CD40 monoclonal antibody (mAb) M3, which completely blocked rhsCD40L-induced platelet activation, also prolonged PFA-100 closure times of normal human blood. In contrast, CD40 mAb G28-5 showed less potential in blocking rhsCD40L-induced CD62P expression and did not affect PFA-100 closure times. However, when added to the platelets after rhsCD40L, G28-5 significantly enhanced the platelet response by causing clustering of, and signaling through, FcgammaRII. Similarly, higher order multimeric immune complexes formed at a 1/3 molar ratio of M90, a CD40L mAb, to rhsCD40L induced strong Fcgamma RII-mediated platelet activation when translocated to the platelet surface in a CD40-dependent manner, including the induction of morphological shape changes, fibrinogen binding, platelet aggregation, dense granule release, microparticle generation and monocyte-platelet-conjugate formation. The results suggest that CD40 may play a role in primary hemostasis and platelet biology by two independent mechanisms: First, by functioning as a primary signaling receptor for CD40L and, second, by serving as a docking molecule for CD40L immune complexes. The latter would also provide a potential mechanistic explanation for the unexpected high incidence of CD40L mAb-associated thrombotic events in recent human and animal studies.

Purification and properties of human melanoma cell tissue factor.

Tissue factor (TF) is a transmembrane glycoprotein that acts as a receptor for nonactivated and activated factor VII (FVII) and triggers the coagulation cascade. TF plays an important role in hemostasis, but may also have noncoagulation functions in vascular development, angiogenesis, and tumor cell metastasis. In tumor cells, analysis of the role of TF has been hampered by the lack of purified TF. In this study, TF antigen was identified on human A375 malignant melanoma cells using flow cytometry. We further purified TF apoprotein 2.000-fold to homogeneity from A375 melanoma cells using immunoaffinity chromatography. On SDS-polyacrylamide gel electrophoresis under reduction, purified TF apoprotein gave two major protein bands corresponding to molecular weights of 53 and 34 to 36 KD. The identity of these forms of TF was confirmed by Western blotting using a polyclonal antibody against human brain TF. Under reduction, the TF antibody bound with a monomeric form of TF (53 KD), and without reduction, to several forms of TF (34 to 128 KD). Preliminary carbohydrate analysis suggested that TF is a glycoprotein and contains about 22% total carbohydrates. The coagulant activity of the purified apoprotein was reconstituted by the addition of phospholipids. The effects of varying concentrations (0 to 8 μg) of polyclonal antibodies to TF and FVII on TF procoagulant activity were studied. Both antibodies inhibited more than 70% of the procoagulant activity of TF in an FX activation assay. The complex formation between purified TF apoprotein and FVlla was demonstrated by using an enzyme-linked immunosorbent assay. TF formed a complex with FVIIa in a concentration-dependent and saturable manner. We conclude that in human melanoma cells, TF occurs in monomeric and heterodimeric forms and appears to have similar properties as reported for TF from other sources.

Purification and some properties of a protein obtained from normal human plasma which inhibits the platelet aggregation induced by thrombotic thrombocytopenic purpura plasma

Plasma from patients with thrombotic thrombocytopenic purpura (TTP) caused the aggregation of washed human platelets, which was inhibited by preincubation with normal plasma. Using salt fractionation, ion exchange chromatography, and preparative agarose gel electrophoresis, we purified a protein from normal plasma which inhibited the platelet aggregation caused by TTP plasma. On SDS polyacrylamide gel, the purified inhibitor gave a single band with a M.W. of 150,000. The antiserum against the purified protein neutralized the activity of the inhibitor and formed an identical precipitin line against normal and TTP plasma.

Electron microscopic study of platelet agglutination induced by thrombotic thrombocytopenic purpura plasma containing 37-KDa platelet agglutinating protein

It has been demonstrated that plasma from a patient with thrombotic thrombocytopenic purpura (TTP) and 37-KDa platelet-agglutinating protein (PAP p37) purified from the same plasma caused the agglutination of platelets from normal subjects as well as from the same patient after recovery without the requirement of extracellular Ca++ and fibrinogen. Experiments were designed to study the morphologic changes of platelets as a result of agglutination and the distribution of platelet receptors for PAP p37 under transmission electron microscope. Following incubation with TTP plasma or PAP p37 with stirring, platelets showed shape change, pseudopod formation, variable degrees of degranulation, dilatation of open canalicular systems and formation of agglutinates composed of a few to several hundred platelets. After platelets were incubated with TTP plasma or PAP p37 they were washed and further incubated with rabbit anti-PAP p37 serum without stirring followed by immuno-staining. Abundant electron dense reaction products were bound directly and randomly to the outer surface of the membrane of solitary platelets. When the reaction mixture was stirred, electron dense particles were also present between the platelet membranes in the agglutinates. No staining was observed in control experiments using normal plasma or non-immune rabbit serum. These results indicate that the TTP plasma containing PAP p37 causes agglutination, shape change, and variable degrees degranulation in platelets and that PAP p37 binds randomly to the outer surface of platelet membrane.