A rapid enzyme-linked assay for ADAMTS-13.

BACKGROUND: A deficiency in the plasma metalloprotease ADAMTS-13 is associated with deposition of microvascular thrombi that cause thrombotic thrombocytopenic purpura. Current assays for ADAMTS-13 are technically complex and time-consuming. The objective of this study is to devise a rapid and sensitive assay for ADAMTS-13 activity in plasma and verify the site of cleavage. METHOD: A new enzyme-linked substrate, which contains a core ADAMTS-13-specific peptide conjugated to horseradish peroxidase (HRP) at the N-terminus, and labeled with biotin at the C-terminus, was constructed. After cleavage of this substrate by plasma ADAMTS-13 and removal of uncleaved substrate by adsorption with streptavidin-agarose, ADAMTS-13 activity was quantitated by determining the unadsorbed HRP activity remaining in solution. Levels of inhibitory antibodies in test plasma were also determined by measuring the residual ADAMTS-13 activity after varying amounts of test plasma were incubated with a known amount of ADAMTS-13. RESULTS: Plasma ADAMTS-13 activity was readily determined in approximately 60 min (coefficient of variation 5.8%) using 1 microL of test plasma. Amino acid sequencing of the cleavage product confirmed that cleavage occurred at the Tyr1605-Met1606 bond in the substrate. ADAMTS-13 activities in the plasma of five TTP patients were below 2%. Inhibitory antibody titers in these samples varied from undetectable to 81 BU mL(-1). CONCLUSION: The HRP-linked substrate provides a rapid, sensitive, and reproducible way of determining the levels of ADAMTS-13 activity and inhibitory antibodies in plasma.

Mutations in a member of the ADAMTS gene family cause thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening systemic illness of abrupt onset and unknown cause. Proteolysis of the blood-clotting protein von Willebrand factor (VWF) observed in normal plasma is decreased in TTP patients. However, the identity of the responsible protease and its role in the pathophysiology of TTP remain unknown. We performed genome-wide linkage analysis in four pedigrees of humans with congenital TTP and mapped the responsible genetic locus to chromosome 9q34. A predicted gene in the identified interval corresponds to a segment of a much larger transcript, identifying a new member of the ADAMTS family of zinc metalloproteinase genes (ADAMTS13). Analysis of patients' genomic DNA identified 12 mutations in the ADAMTS13 gene, accounting for 14 of the 15 disease alleles studied. We show that deficiency of ADAMTS13 is the molecular mechanism responsible for TTP, and suggest that physiologic proteolysis of VWF and/or other ADAMTS13 substrates is required for normal vascular homeostasis.

Thrombotic thrombocytopenic purpura--a syndrome caused by multiple pathogenetic mechanisms.

Platelet thrombus formation in small vessels in thrombotic thrombocytopenic purpura (TTP) is triggered by certain stimuli to cause vascular injury, primary platelet aggregation, or both. The formation and dissolution of platelet thrombi are modulated by proteolysis, plasma factors, PGI2 synthesis and stability, and immune mechanisms. It has been demonstrated that TTP plasmas contain heterogeneous platelet aggregating factors and that TTP plasmas induce the apoptosis of microvascular endothelial cell. Fibrinolysis has been shown decreased. Von Willebrand factor (vWF) under high shear stress is unfolded and becomes adhesive to platelets to induce platelet aggregation. Recently it is found that vWF protease is deficient in hereditary TTP, intermittent relapsing TTP, idiopathic acute TTP and ticlopidine- and clopidogrel-induced TTP, but normal in hemolytic uremic syndrome and organ transplantation-related thrombotic microangiopathy. Apparently absence of vWF protease leaves unfolded adhesive vWF unchecked to cause platelet aggregation. Antibodies against vWF protease are present in intermittent relapsing TTP, idiopathic acute TTP, and ticlopidine-induced TTP. Plasma exchange and/or infusion with or without corticosteroids remain the cornerstone of treatment.

Antibodies to von Willebrand factor-cleaving protease in acute thrombotic thrombocytopenic purpura.

BACKGROUND: Thrombotic thrombocytopenic purpura is a potentially fatal disease characterized by widespread platelet thrombi in the microcirculation. In the normal circulation, von Willebrand factor is cleaved by a plasma protease. We explored the hypothesis that a deficiency of this protease predisposes patients with thrombotic thrombocytopenic purpura to platelet thrombosis. METHODS: We studied the activity of von Willebrand factor-cleaving protease and sought inhibitors of this protease in plasma from patients with acute thrombotic thrombocytopenic purpura, patients with other diseases, and normal control subjects. We also investigated the effect of shear stress on the ristocetin cofactor activity of purified von Willebrand factor in the cryosupernatant fraction of the plasma samples. RESULTS: Thirty-nine samples of plasma from 37 patients with acute thrombotic thrombocytopenic purpura had severe deficiency of von Willebrand factor-cleaving protease. No deficiency was detected in 16 samples of plasma from patients with thrombotic thrombocytopenic purpura in remission or in 74 plasma samples from normal subjects, randomly selected hospitalized patients or outpatients, or patients with hemolysis, thrombocytopenia, or thrombosis from other causes. Inhibitory activity against the protease was detected in 26 of the 39 plasma samples (67 percent) obtained during the acute phase of the disease. The inhibitors were IgG antibodies. Shear stress increased the ristocetin cofactor activity of von Willebrand factor in the cryosupernatant of plasma samples obtained during the acute phase, but decreased the activity in cryosupernatant of plasma from normal subjects. CONCLUSIONS: Inhibitory antibodies against von Willebrand factor-cleaving protease occur in patients with acute thrombotic thrombocytopenic purpura. A deficiency of this protease is likely to have a critical role in the pathogenesis of platelet thrombosis in this disease.

Binding properties and inhibition of platelet aggregation by a monoclonal antibody to CD31 (PECAM-1).

CD31/platelet endothelial cell adhesion molecule 1 (PECAM-1) is expressed on platelets, endothelial cells, myeloid cells, monocytes, and certain lymphocyte subsets. It has been shown that CD31 is involved in the homophilic and heterophilic cellular adhesion and leukocyte transendothelial migration, but little is known about the role of CD31 in platelet functions. Previously we have shown that monoclonal antibody (MAb) AAP2 produced in our laboratory bound to a 110-kD platelet antigen and gave an enhanced binding to activated platelet membrane. In this study we demonstrated that platelet lysate depleted of the antigen through adsorption by an AAP2-solidified affinity column was bound by MAbs against CD62 and CD42 but not by MAb 5.6E against CD31 or AAP2 on the immunoblot. Rabbit antibodies against CD31 completely inhibited the binding of AAP2 to platelets in the flow cytometry analysis. This indicates that AAP2 is specifically against CD31. 125I-labeled AAP2 bound to resting platelets with 5587 +/- 1765 sites/platelet and a Kd of 1 nmol/L and to thrombin-activated platelets with 17,625 +/- 4865 sites/platelet and a Kd of 0.24 nmol/L. Addition of 10 micrograms/mL AAP2 inhibited the aggregation induced by 4 mumol/L ADP by 78.6%, 6 mumol/L epinephrine by 79.4%, 1 microgram/mL collagen by 78.7%, and 0.25 U/mL thrombin by 29%. The platelet aggregation was completely restored when higher concentration of agonists were used. MAb 5.6E did not have any effect on platelet aggregation. These results suggest that AAP2 binds to a special epitope of CD31 on platelets and that CD31 is involved in platelet aggregation.

Purification and characterization of lupus anticoagulant like protein from Agkistrodon halys brevicaudus venom.

By means of CM-Sephadex C-25, DEAE-Sephadex A-50, Sephadex G-200, and Sephadex G-75 chromatographies, a lupus anticoagulant like protein (LALP) from Agkistrodon halys brevicaudus was purified. On SDS-PAGE, the purified LALP had a molecular weight of 25,500 daltons under non-reducing condition and 15,000 daltons under reducing condition. The isoelectric point was pH 5.6. Its N terminal amino acid sequencing revealed a mixture of 2 sequences: DCP(P/S)(D/G)WSSYEGH(C/R)(Q/K). It was devoid of phospholipase A, fibrino(geno)lytic, 5'-nucleotidase, L-amino acid oxidase, phosphomonoesterase, phosphodiesterase and thrombin-like activities, which were found in crude venom. In the presence of LALP, PT, aPTT, and dRVVT of human plasma were markedly prolonged and its effects were concentration-dependent but time-independent. The inhibitory effect of LALP on the plasma clotting time was enhanced by decreasing phospholipid concentration in TTI test. The individual clotting factor activity was not affected by LALP when higher dilutions of LALP-plasma mixture were used for assay. Russell's viper venom time was shortened when high phospholipid confirmatory reagent was used. Therefore, the protein has lupus anticoagulant property.

A monoclonal antibody that recognizes a 110-kDa protein primarily located on the platelet alpha-granule, which is translocated to and upgraded on the surface membrane of platelets upon activation.

A murine monoclonal antibody, designated as MAb AAP2, was produced by immunizing human activated platelets to BALB/c mice. MAb AAP2 recognizes a platelet membrane antigen that was present in trace amount on the membrane of resting platelets, and was strongly expressed on the membrane after activation with thrombin as demonstrated by immunofluorescence microscopy. Platelet granule fractionation by ultracentrifugation on sucrose density gradient and metrizamide showed that the target antigen of MAb AAP2 was located in alpha-granules but not in lysosomes or dense granules. On Western blot prepared with whole platelet lysate, MAb AAP2 bound to a 110-kDa protein under nonreducing and reducing conditions. These results suggest that MAb AAP2 recognizes a 110-kDa platelet antigen, which is primarily located on the alpha granule of platelets and translocated to and upgraded on the surface membrane upon activation.

Purification and characterization of L-amino acid oxidase from king cobra (Ophiophagus hannah) venom and its effects on human platelet aggregation.

Venoms of several snake species contain large amounts of L-amino acid oxidase but its effects on human plasma coagulation and platelet aggregation have not been explored. We have purified L-amino acid oxidase from king cobra venom through CM-Sephadex C-25, Sephadex G-100 and DEAE Sephadex A-50 chromatographies. The purified enzyme has a mol. wt of 135,000 as determined by gel filtration and 65,000 by SDS-PAGE under non-reducing and reducing conditions. Incubation of plasma with L-amino acid oxidase at 200 micrograms/ml did not affect prothrombin time, activated partial thromboplastin time, or thrombin time. Upon addition of L-amino acid oxidase, platelets in platelet-rich plasma were aggregated. The enzyme-induced aggregation was abolished by catalase. The aggregation was also inhibited by indomethacin, aspirin, ethylenediaminetetraacetate, sodium nitroprusside, prostaglandin E1, mepacrine and verapamil, but not by heparin, hirudin, creatine phosphate/creatine phosphokinase or antimycin/2-deoxy-D-glucose. These results suggest that L-amino acid oxidase induces human platelet aggregation through the formation of H2O2, and subsequent thromboxane A2 synthesis requiring Ca2+ but independent of ADP release. The platelet aggregation caused by L-amino acid oxidase is likely to contribute to toxicity inflicted by cobra venom.

Thrombotic microangiopathy and retroviral infections: a 13-year experience.

Eleven of fifty serum samples collected from patients with a diagnosis of thrombotic microangiopathy (TMA), from 1979 to 1991, tested positive for antiretroviral antibodies. Seven had human immunodeficiency virus (HIV) infection, and four had human lymphotrophic virus, type I (HTLV-I) infection. All patients were treated with plasma exchange and/or infusion, but only two of the HIV-infected patients obtained a complete response (CR) and one of them died after a few months. Combined results from the literature indicate that most patients with HIV infection survive less than one year from the initial diagnosis of TMA. In the setting of HIV infection, TMA is a treatable condition, but survival for most patients is less than 12 months. Three of the four HTLV-I infected patients with TMA had a CR. These observations strongly suggest that both HIV and HTLV-I infections are associated with TMA, but rigorous epidemiologic studies will be needed to determine the relative risk for each. Retroviral infections should be considered in patients with TMA, especially if the patient has associated risk factors and demographic characteristics.

Characterization of platelet agglutinating protein p37 purified from the plasma of a patient with thrombotic thrombocytopenic purpura.

We have previously reported the purification of a 37 KDa platelet agglutinating protein (PAP p37) from the plasma of a patient with thrombotic thrombocytopenic purpura and have shown that it is present in a subset of TTP patients, but absent in normal subjects. In this study, we would like to report some of the physico-chemical and immunological properties of this protein. The native molecular weight of PAP p37 from gel filtration was found to be 36,000, which is in agreement with denatured molecular weight (37,000), determined by SDS--polyacrylamide gel electrophoresis under both reducing and non-reducing conditions. The values of Stoke's radius (25A), diffusion coefficient (8.59 x 10(-7)cm2/s) and frictional ratio (1.13), determined by molecular sieve chromatography, suggest that the native protein is compact and globular. The purified protein has an S20,w of 3.5s. Preliminary carbohydrate analysis suggested that p37 is a glycoprotein and contained about 11% neutral sugars and 6.6% sialic acid. Amino acid analysis indicated that the protein is relatively rich in aspartate and serine and has low cysteine, methionine and tryptophan contents. In dot immunobinding ELISA assay, PAP p37 did not react with antibodies to thrombospondin, fibrinogen, fibronectin, plasminogen and von Willebrand factor. Our results suggest that PAP p37 is a single polypeptide compact and globular glycoprotein and is immunologically not related to the aforementioned proteins.

Mechanism of platelet aggregation induced by a monoclonal antibody requiring Fc portion.

A monoclonal antibody designated Apt4, which is IgG1, was produced by fusion of mouse myeloma cells to spleen cells from a BALB/c mouse immunized with normal human platelets. Apt4 whole IgG caused the aggregation of both platelet rich plasma (PRP) and washed platelets from normal subjects and a patient with Bernard Soulier syndrome but not those from two patients with the Type 1 Glanzmann's thrombasthenia. No aggregation was observed when Apt4 F(ab')2 fragments were used. Immunofluorescence study showed that both whole IgG and F(ab')2 fragments of Apt4 bound to fresh or formalin fixed platelets from normal subjects and a patient with Bernard Soulier syndrome but not to those from two patients with Glanzmann's thrombasthenia. Aggregation induced by Apt4 IgG was inhibited by EDTA (10 mM), PGE1 (1 mM), 2-deoxy-D-glucose/antimycin (1.4 uM), and apyrase (20 units/ml). Preincubation of normal PRP with monoclonal anti-GPIIb/IIIa or anti-GPIb antibodies completely or partially inhibited the Apt4-induced aggregation, whereas anti-GPIIIa antibodies have no effects on this activation. Monoclonal ant-Fc gamma RII antibody (IV.3) inhibited Apt4 induced aggregation. Immunoprecipitation of 125I-labeled platelet membrane lysate by Apt4 IgG showed two protein bands with a molecular weight of 145,000 and 95,000 daltons respectively under non-reducing condition, which are corresponding to GPIIb and GPIIIa. In conclusion, Apt4 antibody binds to GPIIb/IIIa complex and induces aggregation, requiring energy metabolism, calcium, ADP release and Fc portion of IgG to interact with Fc receptor, but independent of thromboxane A2 formation.

Platelet-agglutinating protein p37 from a thrombotic thrombocytopenic purpura plasma forms complexes with platelet membrane glycoprotein IV (CD36).

We have previously reported the purification of a 37 kDa platelet agglutinating protein (PAPp37) from the plasma of a patient with Thrombotic Thrombocytopenic purpura (TTP), and have shown recently that p37 causes platelet agglutination through its binding to membrane glycoprotein IV (GPIV). To gain further insight into the mechanism of p37 binding to GPIV, we have studied the interaction between p37 and GPIV. We now demonstrate specific complex formation of p37 with GPIV. In Western immunoblotting p37 binds to purified GPIV and the complex formed between the two proteins was detected by polyclonal antibody to p37 and peroxidase conjugated second antibody. No binding of p37 was noticed with the purified GPIIIa. A solid phase binding assay was developed to study the complex formation. Microtiter wells were coated with GPIV and the control proteins; 125I-p37 was added, allowed to bind and bound radioactivity was measured. Several lines of evidence indicate that the binding of p37 to GPIV was specific. a) GPIV immobilized on Immulon-2 wells bound 10-30 fold more p37 than immobilized fibrinogen, GPIIIa, and BSA. b) Polyclonal antibodies against p37 and GPIV inhibited the binding by 39-68% as compared with control IgG. c) GPIIIa antibody did not inhibit the binding. Molecular sieve chromatography of a mixture of 125I-p37 and GPIV also revealed the fluid phase complex formation ranging in molecular weight from 132,000 to over 350,000 daltons. These results show the specific interaction between p37 and GPIV and suggest that GPIV functions as a p37 receptor during platelet agglutination.

Hepatocellular carcinoma in hemophilia.

A questionnaire-based survey involving 11,801 hemophiliacs from 54 hemophilia centers in the USA and Europe documented the occurrence of hepatocellular carcinoma (HCC) in 10 patients. The crude rate of HCC was 3.2/100,000 patients/year, at least 30 times higher than the background incidence of this tumor in the countries of origin of the patients. All patients were Caucasians with hemophilia A, 39 to 74 years of age, and had liver cirrhosis. All had one or more risk factor for cirrhosis and HCC: 5 were positive for serum hepatitis B surface antigen, 4 had the antibody to hepatitis C virus, and 4 had histories of alcohol abuse. Serum alpha-fetoprotein, measured in 6 patients, was significantly elevated in 4 (range: 807-1399 ng/ml), and only moderately elevated in 2 (25 and 171 ng/ml). The onset of HCC was asymptomatic in 5 patients, whereas it was accompanied by jaundice, abdominal pain, or ascites in the remaining patients. Thus, HCC seems to be a more important secondary disease for hemophiliacs than formerly recognized. Since HCC is often asymptomatic, screening hemophiliacs with chronic liver disease with periodic ultrasound scans might increase the changes of detecting HCC at a stage amenable to surgical treatment.

Platelet agglutinating protein p37 causes platelet agglutination through its binding to membrane glycoprotein IV.

A 37 kDa platelet agglutinating protein (PAP p37) has previously been shown to be present in a subset of patients with thrombotic thrombocytopenic purpura and has been purified from their plasma. Using solubilized platelet membrane proteins from normal donors, it was shown by Western blotting that 125I-p37 bound to a membrane protein of 97 kDa (red/unred). Furthermore, the same protein was identified by reverse immunoblotting in which purified p37 was electrophoresed, transferred to the nitrocellulose sheet and incubated with solubilized normal platelet membrane proteins. The complex formed between p37 and the membrane protein was identified by autoradiography using polyclonal and monoclonal (OKM5) anti-GPIV antibodies, but was not detected by polyclonal antibody to GPIIIa. Similar studies with purified platelet GPIV under both reducing and non-reducing conditions demonstrated the binding of 125I-p37. Polyclonal and monoclonal antibodies to GPIV completely inhibited the platelet agglutination induced by TTP plasma containing p37, however, normal rabbit IgG, rabbit anti-GPIIIa IgG, and murine monoclonal anti-GPIIb/IIIa (10E5) antibodies had no effect. These data indicate that platelet GPIV is the receptor site for PAP p37.

Binding of platelet agglutinating protein p37 from the plasma of a patient with thrombotic thrombocytopenic purpura to human platelets.

We have previously reported the purification of a 37 kDa platelet agglutinating protein (PAP p37) from the plasma of a patient with thrombotic thrombocytopenic purpura (TTP) that was shown to be present in a subset of TTP patients. To gain further insight into the interaction between PAP p37 and platelets, we have studied the properties of PAP p37 binding to platelets. Washed human platelets from two normal donors and two TTP patients after recovery were used for the experiments. The PAP p37 binding to platelets was specific, concentration dependent and saturable. Scatchard analysis demonstrated about 20,564-27,090 PAP p37 binding sites per platelet. Stimulation of platelets with thrombin or ADP did not have any significant effect on its binding. Thiol- and serine-specific protease inhibitors did not inhibit PAP p37 binding to the platelets. Sugars such as glucose, fructose, mannose, and sialic acid, at 40 mM, inhibited its binding to platelets by 44%, 73%, 79%, and 91% respectively, but galactose and amino sugars did not have any significant effect. At 250 micrograms/ml, Concanavalin-A inhibited 42% of binding, but other lectins, such as phytohemagglutinin-P, potato lectin and helix pomatia lectin (snail), did not. Pre-incubation of 125I-PAP p37 with the adult human IgG, decreased its binding to the platelets. The monoclonal antibodies to GP Ib (6D1) and GP IIb-IIIa complex (10E5) did not inhibit the binding of 125I-PAP p37 to platelets. Fibrinogen and von Willebrand factor did not affect the binding either. These results suggest that PAP p37 binds to platelets on the sites other than GP Ib or GP IIb-IIIa complex.

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.

Combination immunosuppressive therapy after factor VIII infusion for acquired factor VIII inhibitor.

STUDY OBJECTIVE: To evaluate the effectiveness of combined cyclophosphamide, vincristine, and prednisone (CVP) therapy after antigenic stimulation with factor VIII in the eradication of factor VIII inhibitor. DESIGN: Factor VIII activity and inhibitor titer were measured before and after FVIII-CVP therapy and patients with factor VIII inhibitor were followed for at least 2 years. SETTING: The first course of therapy was carried out in the hospital when nonhemophiliac patients were admitted for bleeding. Otherwise, treatment was administered at the outpatient clinic. PATIENTS: From 1975 to 1986 we studied 12 nonhemophiliac and 5 hemophiliac patients with factor VIII inhibitor treated with FVIII-CVP and followed at our clinic. INTERVENTION: Patients were infused with one dose of factor VIII concentrate, 50 to 100 U/kg body weight, followed by cyclophosphamide, 500 mg on day 1 and 200 mg/d on days 2 to 5; vincristine, 2 mg on day 1; and prednisone, 100 mg/d on days 1 to 5. This regimen was repeated every 3 to 4 weeks. RESULTS: Of 12 nonhemophiliac patients, 11 responded after 1 to 3 courses of FVIII-CVP with complete disappearance of the inhibitor without recurrence. Among 5 patients with hemophilia who were given 3 to 8 courses, only 1 patient responded with a transient disappearance of inhibitor. Mild neutropenia and infection occurred in 3 patients and required antibiotic treatment. CONCLUSION: Factor VIII-CVP therapy is highly effective in the eradication of factor VIII inhibitor in nonhemophiliac patients but not in patients with hemophilia.

Mechanism of rabbit platelet agglutination induced by acidic mucopolysaccharide extracted from Stichopus japonicus Selenka.

The effects of acidic mucopolysaccharide extracted from sea cucumber (Stichopus japonicus Selenka) (SJAMP) on rabbit platelets were studied. Using citrated platelet-rich plasma (PRP), washed platelets, and formaldehyde fixed platelets from 10 New Zealand white rabbits, we investigated the effects of platelet inhibitors and various plasma and its fractions on SJAMP-induced agglutination. It was found that the tracing of platelet agglutination induced by SJAMP showed a single phase without a lag period. The lowest concentration of SJAMP required for the agglutination of rabbit platelets was approximately 2 micrograms/ml, and the magnitude of agglutination induced by SJAMP was concentration dependent. In 8 out of 10 rabbits, the platelets in PRP were agglutinated by 10 micrograms/ml of SJAMP. Platelet inhibitors, such as aspirin, indomethacin, apyrase, antimycin, 2-deoxy-D-glucose and EDTA did not inhibit the agglutination induced by SJAMP. Washed rabbit platelets were not agglutinated by SJAMP even though the concentration of SJAMP was raised up to 50 micrograms/ml. When rabbit plasma, serum, or 50-60% ammonium sulfate saturated plasma fraction was added to the reaction mixture, agglutination of washed platelets by SJAMP was recovered completely. But human plasma or fibrinogen did not have any effect on the reactivity of washed rabbit platelets to SJAMP. From these data we conclude that the SJAMP-induced rabbit platelet agglutination is independent of energy metabolism but requires plasma cofactor(s) other than fibrinogen. The plasma cofactor is present in 50-60% ammonium sulfate saturated plasma fraction.