High molecular weight kininogen: localization in the unstimulated and activated platelet and activation by a platelet calpain(s)

High mol wt kininogen (HMWK), the major cofactor-substrate of the contact phase of coagulation, is contained within and secreted by platelets. Studies have been performed to localize platelet HMWK in both the unstimulated and activated platelet and to ascertain the effect of platelet enzymes on HMWK itself. On platelet subcellular fractionation, platelet HMWK was localized to alpha-granules, and platelets from a patient with a deficiency of these granules (gray platelet syndrome) had 28% normal platelet HMWK. Platelet HMWK, in addition to being secreted from the platelet, was also localized to the surface of the platelet when activated. Using a competitive enzyme- linked immunosorbent assay for HMWK as an indirect antibody consumption assay, the external membrane of thrombin-activated platelets as well as the releasate from these stimulated platelets had 17 ng HMWK antigen/10(8) platelets available, whereas unstimulated platelets and their supernatant had only 4.9 and 4.2 ng HMWK/10(8) platelets present, respectively. The anti-HMWK antibody consumption by activated normal platelets was specific for membrane-expressed platelet HMWK, since activated platelets from a patient with total kininogen deficiency did not adsorb the anti-HMWK antibody. Enzymes in the cytosolic fraction of platelets cleaved 125I-HMWK (mol wt 120,000) into a mol wt 100,000 polypeptide as well as smaller products at mol wt 74,000, mol wt 62,000, mol wt 47,000, and a few components below mol wt 45,000. No cleavage products were observed when DFP and leupeptin were present. The cleavage of HMWK was specifically prevented by inhibitors of calcium-activated cysteine proteases (leupeptin, N-ethylmaleimide, iodoacetamide, and EDTA) but not by inhibitors of serine proteases (DFP, benzamidine, soybean trypsin inhibitor, or aprotinin). Platelet cytosol increased the coagulant activity of exogenous purified HMWK with maximum HMWK coagulant activity (35-fold) occurring within ten minutes of exposure to platelet cytosol. Treatment of platelet cytosol with leupeptin prevented the increase in the coagulant activity of exogenous HMWK. These studies indicate that activated platelets express platelet HMWK on their external membrane and platelet enzymes can cleave and increase the coagulant activity of exogenous HMWK.     

Type I Glanzmann thrombasthenia patients from the Iraqi-Jewish and Arab populations in Israel can be differentiated by platelet glycoprotein IIIa immunoblot analysis

A sensitive immunoblot technique for platelet glycoprotein IIIa (GPIIIa) was used to analyze the platelets of patients living in Israel who meet the diagnostic criteria for type I Glanzmann thrombasthenia. When reacted with solubilized normal platelets, a rabbit antiserum to GPIIIa identified a major band at molecular weight (mol wt) 90,000 and three additional minor bands at Mr 110,000, 81,000, and 64,000. The major band could not be detected, and the minor bands were either markedly reduced or absent in the platelet samples from 14 of the 15 patients from the Iraqi-Jewish population. In contrast, in all four Arab patients tested, the major band was detectable, although at markedly reduced levels, and the minor bands were either markedly reduced or absent; an additional minor band at mol wt 47,000 was also present in the platelets from these patients. One Iraqi-Jewish patient had a unique pattern in which two of the bands were present but reduced and two were undetectable. We conclude that the protein defect, and thus presumably the genetic defect, causing Glanzmann thrombasthenia in the majority of patients in the Iraqi-Jewish population differs from that in the Arab population, and we confirm that there is considerable biochemical heterogeneity among the patients who meet the criteria for type I Glanzmann thrombasthenia.     

Spectrin is associated with membrane-bound actin filaments in platelets and is hydrolyzed by the Ca2+-dependent protease during platelet activation

We recently showed that platelets contain submembranous actin filaments that are linked to glycoprotein (GP) Ib on the plasma membrane. In the present study, experiments were performed to determine whether spectrin was associated with these filaments. The membrane-bound filaments were isolated from Triton X-100 (Sigma, St Louis) lysates of unstimulated platelets by differential centrifugation. Platelet spectrin was detected immunologically by using antibodies against human brain and RBC spectrin. Immunoblots showed that platelet spectrin consisted of two polypeptides (mol wt 240,000 and 235,000) that were similar in apparent mol wt to those of the alpha and beta chains of brain spectrin but differed slightly from those of RBC spectrin (mol wt 240,000 and 220,000). Immunoprecipitation experiments identified platelet spectrin as two minor polypeptides migrating on sodium dodecyl sulfate (SDS)- polyacrylamide gels between actin-binding protein (mol wt 250,000) and the platelet polypeptide P235 (mol wt 235,000). Immunoblots of fractions isolated from Triton X-100-lysed platelets revealed that the alpha and beta chains of platelet spectrin were associated almost entirely with the actin filaments that were linked to the plasma membrane. Little spectrin was recovered in the Triton X-100-soluble fraction or with the actin filaments that were not membrane bound. During activation of platelets with thrombin or ionophore A23187, the alpha and beta chains of spectrin were hydrolyzed, generating a major degradation product of mol wt 160,000 and a minor one of mol wt 170,000. These two hydrolytic products were also generated in Triton X- 100 lysates incubated in the presence of Ca2+ but were not produced when lysates were treated with leupeptin, ethylene glycol bis(beta- aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), or N- ethylmaleimide, known inhibitors of the Ca2+-dependent protease. These experiments show that spectrin is a previously unidentified component of the membrane-bound actin filament network and that hydrolysis of spectrin by the Ca2+-dependent protease may regulate the interactions of the filaments during platelet activation.     

Polymorphic glycoprotein-1 on mouse platelets: possible role of Pgp-1 and LFA-1 in antibody-dependent platelet cytotoxicity involving complement

The presence of the Pgp-1 glycoprotein on mouse platelets is demonstrated by antibody-binding techniques, by immunoprecipitation, and by transblotting using the monoclonal antibody (MoAb) C71/26 against Pgp-1. C71/26 immunoprecipitates as a broad band of mol wt 87,000 to 100,000 as determined by radioiodination of the platelet cell surface and by the 3H-sodium borohydride labeling technique. Immunoblotting showed Pgp-1 expression on platelets to be quantitatively similar to its presence on macrophages and resolved platelet Pgp-1 into two bands of mol wt 87,000 and 97,000 whereas Pgp-1 on parasite-elicited peritoneal macrophages showed 82,000 and 87,000 mol wt species. Platelets and monocyte/macrophage cells from either peripheral blood or from the peritoneal cavity showed homogeneous binding of Pgp-1 antibody to greater than 97% of cells by flow cytometry. In contrast, lymphocytes from peripheral blood or from the spleen showed a heterogeneous binding pattern with 20% to 30% of cells being negative, and the majority weakly positive. In functional studies, MoAbs against CR1 and CR3 substantially inhibited platelet immune adherence, whereas C71/26 showed only marginal inhibitor. In contrast, C71/26 and other MoAbs against Pgp-1 inhibited platelet- dependent cytotoxicity of antibody-coated sheep erythrocytes in the presence of C5-deficient mouse plasma whereas M1/70 against CR3 showed no effect. In this assay, MoAbs against the alpha- and beta-subunits of leukocyte functional molecule LFA-1 also inhibited platelet cytotoxicity. These results show that the platelet cell surface moieties Pgp-1 and LFA-1 are involved in or closely associated with antibody-dependent cellular cytotoxicity by platelets.     

Platelet antithrombin defect in malignancy: platelet protein alterations

Sixty-eight patients with malignant disease were divided into two groups based on the results of the platelet antithrombin test (PAT). The normal group had a PAT clotting time ranging from 21.4 to 29.8 seconds, which was equivalent to 25% to 65% inactivation of the 2 U of thrombin added to the test system. The other group showed abnormal PAT clotting time, less than 21.4 seconds or less than 25% thrombin inactivation. The polypeptide composition of platelets from the two patient groups was analyzed by sodium dodecyl sulfate (SDS)- electrophoresis on 7.5% polyacrylamide gels. A polypeptide of 180,000 apparent mol wt was decreased or absent in both Coomassie blue- and Alcian blue-stained gels of the platelets from patients whose PAT was abnormal; this polypeptide comigrated with purified platelet thrombospondin. Tritium labeling of platelet surface glycoproteins by the periodate-borohydride method followed by two-dimensional electrophoresis was performed on platelets of seven patients with abnormal PAT. When they were compared with ten patients with normal PAT, a glycoprotein of 140,000 apparent mol wt with a pl of 4.5 to 5.2 was decreased in platelets of all seven patients with abnormal PAT. Nitrocellulose replicas of one-dimensional gels of platelets from 13 of 14 patients with abnormal PAT showed decreased reaction with an anti- human platelet glycocalicin antiserum. Platelets of these same patients also showed a decreased or absent platelet agglutination induced by ristocetin. Patients with normal PAT had a mean agglutination slope of 1.25 +/- 0.6 (n = 26) as compared with 0.37 +/- 0.34 (n = 26) for the abnormal PAT group (P less than .001). Results indicate that platelets from a subpopulation of tumor patients characterized by decreased platelet antithrombin activity have alterations in two platelet glycoproteins, identified as GPIb and thrombospondin.     

GPIIIa-related PLA1 antigens with different molecular weights: studies in platelets, endothelial cells, and megakaryocytes

The mol wt of the glycoprotein(s) carrying the PLA1 antigen was examined on platelets, megakaryocytes and endothelial cells by immunoblotting with a human polyclonal anti-PLA1 antibody (BE), as well as on four different monoclonal antibodies (MoAbs; DEK-1, DEK-2C, DEK- 10, and DEK-16) raised against GPIIIa, the 100,000-mol wt platelet glycoprotein known to carry the PLA1 antigen. BE reacted with PLA1 positive but not with PLA1 negative platelets. DEK-1 reacted strongly with PLA1 positive platelets but weakly with PLA1 negative platelets. The remaining three MoAbs reacted equally with PLA1 positive as well as negative platelets. BE, DEK-1, DEK-10, and DEK-16 reacted with a 120,000- as well as 100,000-mol wt band on immunoblot of PLA1 positive platelets. The 120,000-mol wt band copurified with affinity purified 100,000-mol wt GPIIIa. Megakaryocytes had a prominent 120,000- as well as 105,000-mol wt band that reacted with BE on immunoblot (the 100,000- mol wt band was not detectable). Umbilical cord endothelial cells from presumed PLA-positive infants had a prominent 100,000-mol wt band that reacted with BE, DEK-16, and DEK-1 (the 120,000-mol wt band was not visualized). The 120,000- and 100,000-mol wt PLA1-positive bands could be digested with proteolytic enzymes to 55,000- to 65,000-mol wt- resistant fragments that retain PLA1 epitopes. Further digestion with endoglycosidase-H lowered the apparent mol wt by approximately 2,000 to 6,000 daltons without affecting PLA1 reactivity. We conclude that the PLA1 antigen is present on a 120,000- as well as 100,000-mol wt glycoprotein of platelets and megakaryocytes, a 105,000-mol wt band of megakaryocytes, and a 100,000-mol wt glycoprotein of endothelial cells. We postulate that the 120,000-mol wt glycoprotein, which shares three or more epitopes with the 100,000-mol wt GPIIIa, may be a post- translational precursor of this species.     

Additional glycoprotein defects in Bernard-Soulier's syndrome: confirmation of genetic basis by parental analysis

The glycoprotein profile of Bernard-Soulier platelets was examined by labeling washed platelets with periodate 3H-sodium borohydride, a procedure that labels greater than 30 glycoproteins on the membrane surface of normal platelets. Three Bernard-Soulier patients were studied; two were siblings and the third was unrelated. The platelet protein and glycoprotein profiles were evaluated under nonreduced and reduced conditions using 5%-15% exponential SDS-polyacrylamide gel electrophoresis. The two siblings completely lacked glycoprotein Ib (GPIb). The unrelated patient had congruent to 7% of the normal level. This was confirmed by two-dimensional nonreduced-reduced SDS- polyacrylamide gel electrophoresis, a procedure that allows clear separation of the disulfide-linked subunits of GPIb, GPIb alpha (mol wt 145,000), and GPIb beta (mol wt 25,000) from other membrane glycoproteins. On one-dimensional analysis, Bernard-Soulier's syndrome (BSS) platelets also lacked the peripheral membrane glycoprotein, GPV (mol wt 82,000) and a low molecular weight glycoprotein, GPIX, (nonreduced or reduced, mol wt congruent to 22,000). The two- dimensional gel system also revealed the absence of a minor glycoprotein with a molecular weight of congruent to 100,000 (GP 100). Quantitation of these proteins solubilized from electrophoretograms showed that the siblings' parents had congruent to 50% levels of GPIb, GPIX, and GP 100. A monoclonal antibody against glycoprotein Ib, FMC 25, was negative by immunofluorescence against Bernard-Soulier platelets and immuneprecipitated both GP Ib and GPIX from Triton X100 solubilized, labeled platelets. The combined results suggest that the apparent genetic absence of multiple proteins in Bernard-Soulier platelets is due, in part, to the presence in normal platelets of a tight membrane complex between glycoprotein Ib and at least one of the other absent glycoproteins.     

Studies with a murine monoclonal antibody that abolishes ristocetin-induced binding of von Willebrand factor to platelets: additional evidence in support of GPIb as a platelet receptor for von Willebrand factor

A murine monoclonal antibody directed at or near a platelet membrane receptor for the von Willebrand factor was produced by the hybridoma technique. Purified F(ab')2 fragments and/or intact antibody completely blocked the agglutination of platelets induced by both ristocetin and bovine von Willebrand factor and the binding of von Willebrand factor antigen to platelets. The antibody also decreased platelet retention, prevented the reduction in platelet electrophoretic mobility caused by bovine von Willebrand factor, and decreased the serum prothrombin time. Radiolabeled F(ab')2 fragments bound to or approximately 2.5 X 10(4) sites on normal platelets with high affinity (KD or approximately 1.5 X 10(-8) M); there was no binding to platelets from 2 patients with the Bernard-Soulier syndrome. Immunoprecipitation and affinity chromatography studies indicated that the antibody binds to glycoprotein lb at a site contained on the externally oriented portion of the GPIb alpha chain (glycocalicin). An unidentified mol wt or approximately 20,000 molecule labeled by periodate/NaB3H4 coprecipitated and copurified with GPIb.     

Thrombasthenia with an abnormal platelet membrane glycoprotein IIb of different molecular weight

We describe an individual with abnormal platelet glycoprotein (GP) IIb of different molecular weight (mol wt), a defect that distinguishes this patient from previously reported thrombasthenics. The patient, a 21-year-old female, has a mild bleeding tendency; her platelets lack adenosine diphosphate (ADP) aggregation and have severely suppressed collagen aggregation but a normal response to ristocetin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of her platelets indicates that they contain two types of GPIIb molecules: one with an abnormal mol wt (122 kd, unreduced; 128 kd, reduced) and one with a normal mol wt (128 kd, unreduced; 118 kd, reduced). Relative to the amount of GPIIb in normal platelets, her platelets contain approximately 35% abnormal GPIIb and 20% normal GPIIb. Fibrinogen binding assays on the patient's platelets indicated that they contained 25% of the normal amount of fibrinogen receptors. Crossed immunoelectrophoresis of the patient's platelets demonstrated the formation of a GPIIb/IIIa complex that was mainly composed of normal mol wt GPIIb and GPIIIa. The patient's father has decreased ADP aggregability, and his platelets also contained both abnormal and normal GPIIb (about 50% of the normal level and about 50% of the normal number of fibrinogen receptors); her mother has only normal GPIIb. These results indicate that the patient has heterozygous GPIIb molecules with an abnormality of GPIIb at the molecular level. Studies on this abnormal GPIIb should provide information about the function of GPIIb and the mechanism of its biosynthesis.     

Platelet dense granule membranes contain both granulophysin and P-selectin (GMP-140).

We recently reported the characterization of a platelet granule membrane protein of molecular weight (mol wt) 40,000 called granulophysin (Gerrard et al: Blood 77:101, 1991), identified by a monoclonal antibody (MoAb D545) raised to purified dense granule membranes. Using immunoelectron-microscopic techniques on frozen thin sections, this protein was localized in resting and thrombin-stimulated platelets. In resting platelets, labeled with antigranulophysin antibodies and immunogold probes, label was localized to the membranes of one or two clear granules per platelet thin section. D545 also labeled dense granules in permeabilized whole platelets and isolated dense granule preparations examined by whole-mount techniques. Expression of granulophysin on the platelet surface paralleled dense granule secretion as measured by 14C-serotonin release under conditions in which lysosomal granule release, as measured by beta-glucuronidase secretion, was less than 5%. After thrombin stimulation, both the surface-connected canalicular system and the plasma membrane were labeled, demonstrating redistribution of granulophysin associated with degranulation. Double labeling experiments with D545 and antibodies to the alpha-granule membrane protein, P-selectin, demonstrated labeling of both P-selectin and granulophysin on dense granule membranes. Distribution of both proteins on the plasma membrane after platelet stimulation was similar. The results demonstrate that granulophysin is localized to the dense granules of platelets and is redistributed to the plasma membrane after platelet activation.     

Posttransfusion purpura associated with an autoantibody directed against a previously undefined platelet antigen

Although alloantibody against the PLA1 platelet antigen is usually found in patients with posttransfusion purpura (PTP), the mechanism of destruction of the patient's own PLA1-negative platelets is unexplained. We used a sensitive immunoblot technique to detect antiplatelet antibodies in a patient with classic PTP. The patient's acute-phase serum contained antibodies against three proteins present in control (PLA1-positive) platelets: an antibody that bound to a previously unrecognized platelet protein of mol wt 120,000 [glycoprotein (GP) 120], antibodies that bound to PLA1 (mol wt 90,000), and an epitope of GP IIb (mol wt 140,000). The antibodies against PLA1 and GP IIb did not react with the patient's own PLA1-negative platelets, control PLA1-negative platelets, or thrombasthenic platelets. In contrast, the antibody against GP 120 recognized this protein in all three platelet preparations, but not in Bernard-Soulier or Leka (Baka)-negative platelets. Antibody against GP 120 was not detected in the patient's recovery serum, although the antibodies against PLA1 and GP IIb persisted. F(ab)2 prepared from the patient's acute-phase serum also bound to GP 120. These results suggest that in PTP, transient autoantibody production may be responsible for autologous (PLA1-negative) platelet destruction. In addition, alloantibodies against more than one platelet alloantigen may be found in this disease. The nature of the GP 120 autoantigen and the GP IIb-related alloantigen defined by our patient's serum remains to be determined.     

Platelet-bound complement (C3) in immune thrombocytopenia

The fixation of complement to the circulating platelet in immune thrombocytopenia was detected by measurement of one of the complement components, C3, on the surface of platelets from patients with idiopathic thrombocytopenic purpura (ITP) and systemic lupus erythematosus (SLE) using the anti-C3 consumption assay. The surface IgG was determined simultaneously using the previously described anti- IgG consumption assay. Washed platelets from normal controls had 3.5 fg (10(-15) g) of C3, or about 11,000 molecules, per platelet, an amount comparable to the IgG (4.1 FG, or 15,000 molecules, per platelet). For most patients with ITP both C3 and IgG were increased on the platelet surface, although for 5 of 16 patients only IgG was increased. Two patients with SLE and thrombocytopenia had an increase in both C3 and Ig, six patients with SLE who were not thrombocytopenic had normal amounts of membrane-bound C3 and IgG. In 5 patients, 3 with ITP and 2 with collagen vascular disease, both surface immunoproteins decreased with successful treatment of the thrombocytopenia.     

Thrombin binding and response in platelets from patients with dyslipoproteinemias: increased stimulus-response coupling in type II hyperlipoproteinemia

Platelets were obtained from patients with various hyperlipidemias [type II, type V, lecithin-cholesterol acyltransferase (LCAT) deficiency] and hypolipidemias (abetalipoproteinemia, Tangier disease) to ascertain relationships among plasma lipids, platelet lipids, thrombin binding and thrombin-induced platelet aggregation, and to compare these data with those previously obtained on stimulus-response coupling in platelets following in vitro modification of membrane microviscosity. Washed platelets were studied for their ability to bind 125I-thrombin in the range of 10(-10) to 10(-6) mol/L (10 mU/mL to 100 U/mL) and to aggregate with thrombin at concentrations less than 10(-9) mol/L (100 mU/mL). The values for binding and aggregation in eight patients from six kindred with familial hypercholesterolemia, taken as a group, fell in the low normal range. If divided into two groups, patients with overt cardiovascular disease bound normal amounts of thrombin but were more responsive to it, whereas patients without overt cardiovascular disease bound lower amounts of thrombin but gave an aggregation response in the normal range. These results suggest that platelet hyperresponsiveness in familial hypercholesterolemia arises from an alteration in the coupling mechanism between thrombin binding and response such that platelets from patients with familial hypercholesterolemia are able to respond with lower receptor occupancy than is the case with normal platelets. Thrombin binding and aggregation were within normal ranges for platelets from abetalipoproteinemia patients (N = 4) and type V hyperlipoproteinemia (N = 2), although in the latter case the response appeared to be less at very low thrombin concentrations (less than 30 mU/mL). Thrombin binding was elevated in Tangier disease (N = 3) but with lower responsiveness at lower thrombin concentrations. Thrombin binding was also elevated in LCAT deficiency (N = 2), and one patient showed increased and another showed decreased aggregation responses. In general, increased plasma cholesterol levels resulted in increased stimulus-response coupling (type II), whereas increased triglyceride levels resulted in decreased coupling (type V, Tangier), and there was no apparent alteration in the coupling mechanism with overall reduction in plasma lipid levels as in abetalipoproteinemia.     

Elevated platelet-associated IgG in SLE patients due to anti-platelet autoantibody: differentiation between autoantibodies and immune complexes by ether elution

Summary The level of platelet-associated IgG (PAIgG) is reported to be elevated in patients with systemic lupus erythematosus (SLE). However. the nature of PAIgG is unclear. We have investigated whether the PAIgG of SLE consists of anti-platelet autoantibodies or immune complexes (IC). The PAIgG values measured by flow cytonietry were elevated in 11/25 patients with SLE. 3/6 SLE patients with thrombocytopenia had a high level of PAIgG (the mean fluorescence intensity >10). We used an ether elution technique to determine whether elevated PAIgG consists of anti-platelet antibodies or IC. Preliminary experiments showed that the eluates prepared from platelets sensitized with anti-HPA-4a antibody reacted with normal platelets. while the eluates prepared from platelets sensitized with heat-aggregated IgG or model IC failed to react with normal platelets. These results indicate that the reactivity of eluates can distinguish between platelet-bound antibody and IC. We applied this technique to analysis of the PAIgG of SLE platelets. The eluates from SLE platelets (the mean fluorescence intensity > 10) reacted with normal platelets. indicating that the PAIgG of SLE platelets has the nature of anti-platelet autoantibodies. Furthermore, we investigated the target antigens which bind PAIgGs of SLE, using the direct immunoprecipitation procedure and modified antigen capture ELISA (MACE). Both methods identified GPIIb/IIIa as the target antigens. We conclude that the ether elution technique can distinguish between anti-platelet antibodies and TC. and that the PAIgGs of SLE with a high PAIgG value and thrombocytopenia have the nature of anti-platelet autoantibodies.     

Thrombotic disease in systemic lupus erythematosus is associated with a maintained systemic platelet activation

Patients with systemic lupus erythematosus (SLE) have an increased risk of thrombosis. Platelet-induced extracellular phosphorylation of plasma proteins suggests that this is due to persistent activation of the platelets. We examined 30 SLE patients (15 with thrombotic disease), 18 non-SLE patients with deep vein thrombosis (DVT) and 50 healthy controls by analysing beta-thromboglobulin, activated factor XI-antithrombin complexes and fibrinogen-bound phosphate. All parameters were elevated in SLE patients, particularly those with thrombosis, but normal in DVT cases and healthy controls. We conclude that thrombotic disease in SLE patients is associated with a persistent systemic platelet activation that may lower the threshold for induction of thrombosis.     

A novel platelet aggregating factor found in a patient with defective collagen-induced platelet aggregation and autoimmune thrombocytopenia

We found a novel platelet aggregating factor in a patient with steroid- responsive immune thrombocytopenic purpura that is associated with defective collagen-induced platelet functions. The aggregating factor and platelet functions were analyzed. The patient, a 58-year-old female, had purpura and prolonged bleeding time despite adequate platelet counts (greater than 140,000/microL) after steroid therapy. The patient's platelets responded normally to all agonists except collagen. Platelet adhesion to collagen fibrils was decreased. The patient's plasma induced irreversible aggregation and ATP release in normal platelet-rich plasma (PRP). This platelet aggregating factor was found in F(ab')2 fragments of the patient's IgG, which caused thromboxane B2 synthesis, elevation of cytoplasmic Ca2+ levels, and phosphorylation of 40 kDa protein in normal platelets. Platelet aggregation by the patient's IgG was inhibited by prostacyclin, dibutyryl cAMP, diltiazem, disodium ethylenediaminetetraacetate, and antimycin A plus iodoacetate, but ADP scavengers, cyclo-oxygenase inhibitors, and heparin had little or no effect. The aggregating activity of the patient's IgG absorbed to and eluted from normal platelets. The patient's Fab fragments did not induce platelet aggregation in eight of ten normal PRP but specifically inhibited aggregation induced by collagen and by the patient's IgG. The major component of an immunoprecipitate made with the patient's IgG from radiolabeled membrane proteins of normal platelet extract had a 62 kDa mol wt, while no such precipitate appeared in extracts of the patient's platelets. These results indicated that platelet aggregation by the patient's IgG was induced by the reaction of an antibody with a specific antigen on the normal platelet membrane through stimulus- response coupling. This antigen may be a collagen receptor on the platelet, most likely a polypeptide of 62 kDa under reducing condition. The defect of collagen-induced aggregation of the patient's platelets seemed to be due to alteration of the membrane protein related to this putative collagen receptor.     

Splenic platelet kinetics in systemic lupus erythematosus (SLE)

The splenic blood flow, intrasplenic platelet kinetics and spleen size were determined in 8 females with systemic lupus erythematosus (SLE), all without signs of active disease, by using gamma-camera scintigraphy with 111In-labelled platelets and 99mTc-stannous colloid. The results for splenic blood flow, intrasplenic platelet transit time and splenic platelet pool size, obtained by compartmental analysis of the initial distribution of radiolabelled platelets between blood and spleen, did not differ from those of a control group. In all SLE patients the spleen size was within normal limits. There was a significant relationship between the spleen volume and the splenic platelet pool size (r = 0.75; p less than 0.05), and between the spleen volume and splenic blood flow (r = 0.76; p less than 0.05). A borderline, inverse correlation was present between an estimate of splenic perfusion and intrasplenic platelet transit time (r = 0.62; p = 0.1). It is concluded that the splenic function, measured as splenic blood flow and intrasplenic platelet kinetics, is not disturbed in SLE patients without active disease.     

Identification of and partial characterization of platelet vitronectin: evidence for complex formation with platelet-derived plasminogen activator inhibitor-1

Vitronectin (VN; = complement S-protein), a plasma glycoprotein that is also associated with extracellular sites, was identified in washed human platelets contaminated with less than 0.05% of plasma VN. A specific enzyme-linked immunosorbent assay (ELISA) for VN has been developed and was used to detect and to quantitate VN in detergent extracts of washed platelets with 8.1 +/- 4.6 micrograms/10(9) platelets (n = 10), representing about 0.8% of the plasma VN pool. Platelet and plasma VN were similar by immunochemical criteria using Western-blot analysis, although platelet VN was mainly found as partially proteolyzed polypeptide. Total release of platelet VN occurred at optimal doses of Ca-ionophore 23187 or thrombin, whereas no VN was released by platelet treatment with digitonin or Staphylococcus alpha-toxin. During stimulation of washed platelets with various concentrations of thrombin, the nearly concomitant release of VN and plasminogen activator inhibitor-1 (PAI-1) together with platelet factor 4 indicated the association of VN with inner-platelet storage granules. Furthermore, platelet VN and PAI-1 in Ca-ionophore releasates comigrated during ultracentrifugation in high mol wt fractions of sucrose density gradients, indicating a possible association of both components. Complex formation of platelet VN and PAI-1 was verified by a sensitive enzyme-linked immunosorbent assay (ELISA) and accounts at least in part for a high molecular form of platelet VN. The identification of platelet VN and its binding to platelet PAI-1 raises the possibility that VN, in contrast to other adhesive proteins, may participate in localized regulatory functions of blood coagulation and fibrinolysis in platelet-matrix interactions and the protection of the matrix against proteolysis.     

A rapid quantitation of platelet-associated IgG by nephelometry

Platelet-associated IgG (PAIgG) was measured by a simple rapid nephelometric technique using washed solubilized platelets and commercially available, prestandardized reagents. Normal subjects with normal platelet counts had PAIgG levels of 2.1-6.7 fg/platelet. Subjects with idiopathic immune thrombocytopenic purpura (ITP) had levels of 7.2-43.3 fg/platelet. Ninety percent of ITP patients had values exceeding 2 SD units of the mean of normal subjects. Elevated values were also found in 17% of patients with recovered ITP, patients with SLE with and without thrombocytopenia, patients with thrombocytopenia occurring during septicemia, and patients with IGg myeloma. Results can be obtained within several hours of receipt of blood specimen, and are similar to the reports that used more complex techniques.     

Evaluation of platelet surface antigens: localization of the PlA1 alloantigen

An approach is described for localizing antigenic determinants to specific platelet proteins. Platelets are solubilized, electrophoresed and transferred to nitrocellulose paper where they are immobilized. After incubation with radiolabelled specific antibody, the antigenic determinants are localized by radioautography. Purified proteins ranging in size from 50000 to 540000 could be studied in this manner and still retain their antigenicity. Solubilized immobilized platelet proteins were reacted with serum IgG from two patients with post-transfusion purpura known to have anti-PlA1 antibodies. The radioactivity was associated with a major protein band with an apparent molecular weight of 100000 daltons. However, when purified anti-PlA1 antibody eluted from PlA1 (+) platelets was used, at least six additional minor bands were noted with molecular weights ranging from about 86000 to 175000. No radioactive bands were noted using PlA1 negative platelets or reduced PlA1 (+) platelet proteins. Reaction of the antibody from one patient wih proteins from thrombasthenic platelets resulted in a marked reduction in the intensity of all radioactive bands. In addition, a new high molecular weight band was noted which was not present in reactions containing PlA1 (+) platelets. These data suggest that the PlA1 antigen is primarily but not entirely localized to glycoprotein IIIa. This approach is relatively simple, requires only minimal amounts of antibody and should be generally useful in the study of cellular antigens and other complex mixtures of proteins.