Masaaki Moroi

Abnormal Plasminogen: A HEREDITARY MOLECULAR ABNORMALITY FOUND IN A PATIENT WITH RECURRENT THROMBOSIS

A patient who suffered a recurring thrombosis over the last 15 yr has been investigated. The only abnormality found in this patient was a significantly depressed level of plasminogen activity in plasma. In spite of the depressed plasminogen activity, the patient was found to have a normal level of plasminogen antigen concentration. It was calculated that the activity per milligram of plasminogen of the patient was approximately one-half the values of normal subjects. The same discrepancy between biological activity and antigen concentration was found in the other members of the kindred. A niece was found to have practically no plasminogen activity but possessed a normal concentration of plasminogen antigen. Both her parents were found to have approximately half the normal plasminogen activity and normal antigen levels. These studies suggested that the molecular abnormality was inherited as an autosomal characteristic, and the family members who had half the normal levels of activity with normal plasminogen antigen were heterozygotes whereas the one with practically no plasminogen activity was homozygote. Subsequent studies showed that the pattern of gel electrofocusing of purified plasminogen of the heterozygotes consisted of 10 normal bands and 10 additional abnormal bands, each of which had a slightly higher isoelectric point than each corresponding normal component. This indicates that plasminogen of the heterozygote is a mixture of normal and abnormal molecules in an approximately equal amount, which was substantiated by active site titration of purified plasminogen preparations obtained from the propositus and a normal individual. The gel electrofocusing pattern of the homozygote consisted of abnormal bands only. The defect is a hereditary abnormality of plasminogen.

Platelets with 10% of the normal amount of glycoprotein VI have an impaired response to collagen that results in a mild bleeding tendency

Platelet glycoprotein VI (GPVI), a 62kD membrane protein, has been identified as one of the platelet receptors for collagen, since GPVI-deficient platelets exhibit abnormal responses to collagen and an abnormal bleeding tendency. We report a female patient with a mild bleeding history whose platelets expressed 10% GPVI of normal platelets. Shape change, aggregation and ATP release of the patients platelets were completely absent in response to 1-5 micrograms/ml collagen but present normally in response to ADP and Ca2+ ionophore A23187. Adhesion of the patients platelets to coated collagen was mildly affected (40-60% of normal platelets) in spite of only 10% expression of GPVI. Flow cytometrical studies revealed that the patients platelets expressed normal amounts of the GPIa/IIa complex. These results suggest that platelet GPVI is less involved in adhesion to collagen than shape change and aggregation induced by collagen.

Platelets Interact with Soluble and Insoluble Collagens through Characteristically Different Reactions

Platelet interaction with soluble and insoluble collagens was characterized through binding studies. In contrast to resting platelets, cells reacted with activators, TS2/16 (integrin α2β1-activating antibody), thrombin, collagen-related peptide, or ADP, exhibited specific soluble collagen binding that is Mg2+-dependent, but inhibited by prostaglandin I2, Ca2+, and Gi9 (anti-integrin α2β1 antibody). Each platelet has 1500–3500 soluble collagen binding sites, with a dissociation constant of 3.5–9 × 10−8 m. This is the first study to show the specific binding of soluble collagen to platelets; our data strongly suggest that the receptor is integrin α2β1 after it becomes activated upon platelet activation. These results suggest that activation of platelets transforms integrin α2β1 to a state with higher affinity binding sites for soluble collagen. The soluble collagen-platelet interaction was compared with the platelet interaction with fibrillar collagen, which has until now not been demonstrated to bind specifically to platelets. Here, we demonstrated specific, biphasic fibrillar collagen binding. One phase is rapid and metal ion-independent, and accounts for most of the binding. The other phase is slow and Mg2+-dependent. The characteristic differences in the specific bindings of soluble and fibrous collagens demonstrate the different contributions of two different collagen receptors.

The behavior of alpha2-plasmin inhibitor in fibrinolytic states.

Human plasma alpha2-plasmin inhibitor in fibrinolytic states was studied using immunochemical methods and radioiodinated plasminogen. The concentration and activity of plasma alpha2-plasmin inhibitor decreased when urokinase was added to plasma in vitro or infused intravenously in man. The decrease was associated with the appearance of plasmin-alpha2-plasmin inhibitor complex which subsequently disappeared from the circulation in a short time. A decrease of other major inhibitors, such as alpha2-macroglobulin and alpha1-antitrypsin, was not observed when the amount of urokinase added or infused was relatively small, and conversion of plasminogen to plasmin was not extensive. The formation of plasmin-alpha2-macroglobulin complex was observed only when plasma plasminogen was activated with a larger amount of urokinase, and after most of the alpha2-plasmin inhibitor was consumed by forming complexes with plasmin. The formation of plasmin-alpha1-antitrypsin complex was not observed even in the highly activated plasma unless exogenous plasmin was added to the plasma. alpha2-Plasmin inhibitor was the only inhibitor of which the concentration in plasma was significantly decreased in patients with disseminated intravascular coagulation and fibrinolysis among the major plasmin inhibitors in plasma. The most reactive inhibitor for regulating plasma fibrinolysis very likely is alpha2-plasmin inhibitor.

Activation of the Platelet Collagen Receptor Integrin α2β1: Its Mechanism and Participation in the Physiological Functions of Platelets

When platelets are stimulated by agonists, integrin alpha(2)beta(1) (GP Ia/IIa), one of the platelet collagen receptors, is activated to forms with high affinities for its ligand collagen. Here we describe our studies to characterize the binding kinetics of the activated integrin forms and the activation mechanism. Under low agonist concentrations, integrin alpha(2)beta(1) is activated through a mechanism involving ADP/ADP receptors; and under high agonist concentrations, multiple signaling pathways are involved in its activation. Such differences in mechanism at low and high agonist concentrations are also suggested in the activation of integrin alpha(IIb)beta(3), the platelet fibrinogen receptor. We describe our flow adhesion studies, from which evidence was obtained about the involvement of integrin alpha(2)beta(1) activation in the physiological function of platelets, adhesion and thrombus formation.

Measuring soluble platelet glycoprotein VI in human plasma by ELISA.

Recent experimental evidence demonstrates that the platelet-specific collagen receptor, glycoprotein (GP)VI is essentially all uncleaved on normal circulating platelets, but is shed from the platelet surface in a metalloproteinase-dependent manner in response to GPVI ligands (including collagen), anti-GPVI antibodies or activation at the platelet Fc receptor, FcγRIIa. This raises the question of whether shed ectodomain fragment in plasma could be a useful biomarker of thrombotic risk and/or autoimmune thrombocytopenia. In this study, we developed a sandwich enzyme-linked immunosorbent assay (ELISA) for measuring soluble GPVI in human plasma, using rabbit anti-GPVI polyclonal antibody in the solid-phase, murine anti-GPVI monoclonal antibody (1A12) in the fluid-phase and horseradish peroxidase (HRP)-coupled anti-mouse antibody and enhanced chemiluminescence (ECL) for detection. The ELISA was optimized for sensitivity, reproducibility, inter- and intra-assay precision, addition and recovery and detected GPVI in plasma with a lower detection limit of ∼1 ng/mL. Effects of different anti-coagulants (trisodium citrate, acid-citrate-dextrose or EDTA) were negligible. In ten healthy donors, soluble plasma GPVI levels were 18.9 ± 4.1 ng/mL. Treating normal platelet-rich plasma with a GPVI ligand (collagen-related peptide, CRP), calmodulin inhibitor W7 (that induces GPVI shedding without platelet activation) or N-ethylmaleimide (that directly activates platelet sheddases), under conditions previously shown to induce GPVI shedding, also increased plasma GPVI levels by up to ∼7-fold, compared to previously reported autoimmune (anti-GPVI) patient plasma where soluble GPVI was ∼10-fold higher than normal. Characterization of this sensitive ELISA should facilitate analysis of functional/diagnostic role(s) for soluble GPVI in human plasma associated with thrombotic/immune dysfunction.

Compromised ITAM‐based platelet receptor function in a patient with immune thrombocytopenic purpura

Summary.  Background: Receptors on platelets that contain immunoreceptor tyrosine‐based activation motifs (ITAMs) include collagen receptor glycoprotein (GP) VI, and FcγRIIa, a low affinity receptor for immunoglobulin (Ig) G. Objectives: We examined the function of GPVI and FcγRIIa in a patient diagnosed with immune thrombocytopenic purpura (ITP) who had unexplained pathological bruising despite normalization of the platelet count with treatment. Methods and Results: Patient platelets aggregated normally in response to ADP, arachadonic acid and epinephrine, but not to GPVI agonists, collagen or collagen‐related peptide, or to FcγRII‐activating monoclonal antibody (mAb) 8.26, suggesting ITAM receptor dysfunction. Plasma contained an anti‐GPVI antibody by MAIPA and aggregated normal platelets. Aggregating activity was partially (∼60%) blocked by FcγRIIa‐blocking antibody, IV.3, and completely blocked by soluble GPVI ectodomain. Full‐length GPVI on the patient platelet surface was reduced to ∼10% of normal levels, and a ∼10‐kDa GPVI cytoplasmic tail remnant and cleaved FcγRIIa were detectable by western blot, indicating platelet receptor proteolysis. Plasma from the patient contained ∼150 ng mL−1 soluble GPVI by ELISA (normal plasma, ∼15 ng mL−1) and IgG purified from patient plasma caused FcγRIIa‐mediated, EDTA‐sensitive cleavage of both GPVI and FcγRIIa on normal platelets. Conclusions: In ITP patients, platelet autoantibodies can curtail platelet receptor function. Platelet ITAM receptor dysfunction may contribute to the increased bleeding phenotype observed in some patients with ITP.

Cloning and Expression of the Platelet-Specific Collagen Receptor Glycoprotein VI

Platelet glycoprotein VI (GP VI) was purified from platelet membranes and its internal amino acid sequences were determined. The cloned cDNA of GP VI indicates an open reading frame coding for 20 amino acid signal sequences and a mature protein of 319 amino acids. Its extracellular region has two Ig-like domains and a mucin-like, Ser/Thr-rich region, suggesting that GP VI is a member of the paired Ig-like receptor family. GP VI-transfected cells contained convulxin-(reactive) and antibody against recombinant GP VI-reactive protein bands that migrated at the same position as platelet GP VI in SDS/PAGE-electroblotting. These data indicate that the protein deduced from the cloned cDNA corresponds to platelet GP VI.

INTEGRIN-MEDIATED PLATELET ADHESION

Adhesion of platelets to the damaged subendothelium is a prerequisite reaction for the initiation of hemostasis in vivo. Platelet membranes contain high concentrations of integrins and other glycoproteins (GPs) that are involved in the platelet adhesion to the extracellular matrix. In the present review, we focus on two platelet integrins, integrin alphaIIb beta3 (GPIIb/IIIa) and integrin alpha2 beta1 (GPIa/IIa) because these integrins are major components of the platelet membrane proteins and are known to contribute to platelet adhesion to fibrin(ogen) and collagen surfaces, respectively. These integrins bind soluble ligands (fibrinogen or collagen) after platelets are activated but only have low affinity towards these ligands when platelets are in the resting state. We describe the binding properties of these integrins and discuss the mechanism for the activation of these integrins. Platelets can adhere to fibrin(ogen) or collagen immobilized on a surface. When platelets adhere to a collagen- or fibrin-coated surface, they become activated and form aggregates; this is especially prominent under flow conditions. We discuss the contribution of these integrins and non-integrin proteins, GPIb and GPVI, to the platelet adhesion on to the collagen surface, especially under flow conditions, a system that most closely approximates platelet adhesion in vivo.

Glycoprotein (GP) VI dimer as a major collagen-binding site of native platelets: direct evidence obtained with dimeric GPVI-specific Fabs

Summary.  Background: The platelet collagen receptor glycoprotein (GP) VI is suggested to exist as a dimer on the platelet surface, but no direct proof of the functional importance of dimer formation has been provided. Objectives: To obtain direct evidence for GPVI dimers on the platelet membrane and their functional importance, Fab antibodies were developed that bind to GPVI dimer (GPVI‐Fc2) but not to GPVI monomer (GPVIex) through a phage display method. Results: Ssix Fabs were found: B–F, only reactive with GPVI‐Fc2, and A, mainly reactive with GPVI‐Fc2, with some reactivity towards GPVIex; each Fab (Fab‐dHLX‐MH) forms a bivalent dimer (b‐Fab) by dimerizing the dHLX domains from two Fab molecules. Fab F was subcloned to a monovalent format by deleting its dHLX domain. All b‐Fabs induced platelet aggregation, but the monomeric form of Fab F (m‐Fab‐F) specifically inhibited collagen‐induced aggregation. All b‐Fabs and m‐Fab‐F inhibited GPVI‐Fc2 binding to fibrous collagen. Immunoblotting showed that b‐Fab‐F and m‐Fab‐F bound weakly to GPVI‐Fc2. Adding the anti‐GPVI monoclonal antibody 204‐11 increased the Bmax of m‐Fab‐F binding to GPVI‐Fc2, suggesting that 204‐11 binds to GPVI‐Fc2 molecules not already in the appropriate conformation to recognize the Fab, converting them to a conformation reactive to the Fab. Conclusions: GPVI forms a specific structure by dimerization that is necessary for the binding of this receptor to collagen fibrils. The binding of m‐Fab‐F to platelets directly demonstrates that GPVI is present as a functionally relevant dimer on the platelet surface.