calpain and Thrombocytopenia

Topics: Antibodies; Blood Platelets; Calpain; Endopeptidases; Factor VIII; Glycoproteins; Hemorrhagic Disorders; Humans; Membrane Proteins; Platelet Adhesiveness; Platelet Aggregation; Platelet Glycoprotein GPIb-IX Complex; Platelet Membrane Glycoproteins; Quinidine; Quinine; Receptors, Cell Surface; Receptors, Immunologic; Receptors, Thrombin; Syndrome; Thrombocytopenia

Collagen binding to glycoprotein VI (GPVI) induces signals critical for platelet activation in thrombosis. Both ligand-induced GPVI signaling through its coassociated Fc-receptor gamma-chain (FcRgamma) immunoreceptor tyrosine-activation motif (ITAM) and the calmodulin inhibitor, W7, dissociate calmodulin from GPVI and induce metalloproteinase-mediated GPVI ectodomain shedding. We investigated whether signaling by another ITAM-bearing receptor on platelets, FcgammaRIIa, also down-regulates GPVI expression. Agonists that signal through FcgammaRIIa, the mAbs VM58 or 14A2, potently induced GPVI shedding, inhibitable by the metalloproteinase inhibitor, GM6001. Unexpectedly, FcgammaRIIa also underwent rapid proteolysis in platelets treated with agonists for FcgammaRIIa (VM58/14A2) or GPVI/FcRgamma (the snake toxin, convulxin), generating an approximate 30-kDa fragment. Immunoprecipitation/pull-down experiments showed that FcgammaRIIa also bound calmodulin and W7 induced FcgammaRIIa cleavage. However, unlike GPVI, the approximate 30-kDa FcgammaRIIa fragment remained platelet associated, and proteolysis was unaffected by GM6001 but was inhibited by a membrane-permeable calpain inhibitor, E64d; consistent with this, micro-calpain cleaved an FcgammaRIIa tail-fusion protein at (222)Lys/(223)Ala and (230)Gly/(231)Arg, upstream of the ITAM domain. These findings suggest simultaneous activation of distinct extracellular (metalloproteinase-mediated) and intracellular (calpain-mediated) proteolytic pathways irreversibly inactivating platelet GPVI/FcRgamma and FcgammaRIIa, respectively. Activation of both pathways was observed with immunoglobulin from patients with heparin-induced thrombocytopenia (HIT), suggesting novel mechanisms for platelet dysfunction by FcgammaRIIa after immunologic insult.

Topics: Amino Acid Sequence; Antigens, CD; Blood Platelets; Calmodulin; Calpain; Enzyme Inhibitors; Humans; Immunoglobulins; Ligands; Metalloproteases; Molecular Sequence Data; Platelet Activation; Platelet Membrane Glycoproteins; Protein Structure, Tertiary; Receptors, IgG; Signal Transduction; Sulfonamides; Thrombocytopenia

Platelet activation by potent, Ca(2+)-mobilizing agonists results in shedding of microparticles that are active in coagulation. Here we show that platelets under storage produce procoagulant microparticles in the absence of agonist. Microparticle formation by resting platelets results from alphaIIbbeta3 signaling to destabilization of the actin cytoskeleton in the absence of calpain activation. Integrin-mediated spreading of platelets over fibrinogen similarly results in microparticle formation. After transfusion of stored platelet preparations to thrombocytopenic patients, the microparticles contribute to coagulant activity in vivo.

Topics: Actin Cytoskeleton; Blood Coagulation; Blood Component Transfusion; Blood Platelets; Calcium Channel Blockers; Calpain; Coagulants; Enzyme Activation; Humans; Integrin beta3; Platelet Activation; Platelet Membrane Glycoprotein IIb; Preservation, Biological; Signal Transduction; Thrombocytopenia

The Wiskott-Aldrich syndrome (WAS) is an inherited platelet/T-lymphocyte disease characterized by small platelets, thrombocytopenia and immunodeficiency. Because degradative events have a significant role, we directly examined calpain (Ca(2+)-dependent neutral protease), a prominent protease in the affected cells, by functional and antigenic quantitation. Calpain activity in platelets of seven WAS patients was decreased to 59 +/- 3.7% (P < 0.01) relative to platelets of 11 normals. Platelets of two patients with immune thrombocytopenia had normal calpain activity. By immunoblotting, mu-procalpain, the mu-calpain species in resting (unstimulated) blood cells, was decreased in platelets of nine WAS patients to 58 +/- 14.6% (P < 0.01) relative to paired normals. In contrast, mu-procalpain levels in lymphocytes of seven WAS patients did not differ from normal lymphocytes. Normal platelets and lymphocytes have different mechanisms for Ca(2+)-dependent mu-procalpain activation. On addition of ionophore and Ca2+ to stirred platelets, 80kD mu-procalpain was rapidly (0.5 min) and quantitatively converted to 76 kD active mu-calpain; this process was the same in WAS platelets. In lymphocytes, mu-procalpain activation was slow, only partially complete (40 min), and the active species was 78 kD. The marked depletion of calpain in WAS platelets demonstrated in this study may result from inappropriate stimulation of platelets and be related to the severe thrombocytopenia that characterizes this disease.

Topics: Autoimmune Diseases; Blood Platelets; Calcimycin; Calcium; Calpain; Enzyme Precursors; Humans; Immunoblotting; Lymphocytes; Male; Thrombocytopenia; Wiskott-Aldrich Syndrome