Biomedical subjects
S Karpatkin
Publications and source records attributed to S Karpatkin.
Idiopathic thrombocytopenic purpura in homosexual men.
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Studies of the origin of platelet-associated fibrinogen.
Platelet membranes and whole platelet preparations were examined by crossed immunoelectrophoresis in normal individuals, in a patient with congenital afibrinogenemia, and in two unrelated patients with Glanzmann's thrombasthenia (types I and II) to study the origin of platelet-associated fibrinogen. Results indicate: (1) platelet and plasma fibrinogen are probably derived from the same gene product, (2) platelet fibrinogen is not derived from the surrounding plasma milieu, (3) under basal conditions, platelet fibrinogen is located only within the alpha-granules and not on the platelet surface, (4) addition of trypsin to fibrinogen uncovers either a neoantigen or a hidden pool of platelet fibrinogen in the alpha-granules, (5) platelets from two patients with Glanzmann's thrombasthenia contain near-normal quantities of fibrinogen.
Leukocyte migration inhibition of buffy coats from patients with autoimmune thrombocytopenic purpura when exposed to normal platelets: modulation by transfer factor.
Cellular-mediated immunity was studied in autoimmune thrombocytopenic purpura (ATP) patients by investigating leukocyte migration inhibition (LMI) following the interaction of normal platelets with patients' lymphocytes. When normal platelets were incubated with leukocyte buffy coats of ATP patients, the migration index (MI) was significantly impaired compared to buffy coats from normal subjects, employing 4 different concentrations of platelets. At the highest platelet concentration (10(9)/ml), MI was 0.87 +/- 0.04 (SEM) for ATP lymphocytes compared to 1.05 +/- 0.05 (p less than 0.01) for normal lymphocytes. Nine of 21 patients had an MI less than 0.80, whereas all control subjects had MIs greater than 0.85. Similar results were obtained at 2 different platelet membrane concentrations. At 500 micrograms/ml, the MI for ATP lymphocytes was 0.74 +/- 0.04, compared to 0.98 +/- 0.08 (p less than 0.01) for normal lymphocytes (12 experiments). An inverse relationship was noted between platelet count and lymphokine production in ATP patients (r = 0.815, p less than 0.001, 10 experiments). Transfer factor from an ATP patient in remission converted an abnormal LMI response of 0.68 +/- 0.04 from a patient with severe thrombocytopenia to 0.84 +/- 0.07 (p less than 0.005, 8 experiments). Similar results were obtained with transfer factor from 2 other patients in remission. Transfer factor from a patient with severe thrombocytopenia converted a normal response of 1.04 +/- 0.05 of normal subjects to a lower response of 0.88 +/- 0.04 (p less than 0.03, 12 experiments). Thus, lymphocytes of ATP patients are primed to recognize and be perturbed by normal platelets, whereas normal lymphocytes are not. This indicates specificity of the antigen-lymphocyte reaction in ATP patients. Transfer factor is capable of modulating this response in vitro.
Lack of effect of in vivo prostacyclin on the development of pulmonary metastases in mice following intravenous injection of CT26 colon carcinoma, Lewis lung carcinoma, or B16 amelanotic melanoma cells.
Honn et al. [Science (Wash. DC), 212: 1270, 1981] have recently reported a 93% reduction in the development of metastases of B16 amelanotic tumor cells given i.v. following a single dose of prostacyclin (PGI2) (100 micrograms) and theophylline (100 micrograms) 30 min prior to the injection of tumor cells. We have been unable to reduce pulmonary metastases induced by the i.v. injection of CT26 colon adenocarcinoma, Lewis lung carcinoma, or B16 amelanotic melanoma cells with a similar regimen. Thus, PGI2 and theophylline given prior to injection of tumor cells and 2 hr postinjection had no effect on the number or volume of pulmonary tumor nodules for CT26 cells, using 15 experimental and 14 control animals; Lewis lung cells, using 14 experimental and 13 control animals; or B16 amelanotic cells, using 26 experimental and 12 control animals. The PGI2 used was shown to be active in vitro, inhibiting tumor-induced platelet aggregation by all three tumors at 10(-9)M; and in vivo by inhibition of Lewis lung-induced thrombocytopenia at 1 hr, using 100 micrograms PGI2 prior to the injection of tumor cells.
Effect of antiplatelet antibody on the development of pulmonary metastases following injection of CT26 colon adenocarcinoma, Lewis lung carcinoma, and B16 amelanotic melanoma tumor cells into mice.
Three different murine tumors, CT26 colon adenocarcinoma, Lewis lung carcinoma, and B16 amelanotic melanoma, were injected into syngeneic mice (BALB/c and C57BL/6J) to test the effect of rabbit anti-mouse platelet antibody on the development of pulmonary metastases. Antiplatelet antibody, when injected i.p., decreased the platelet count from 1.5 x 10(6)/microliters to 0.12 x 10(6)/microliters at 6 hr, which remained at this level for 24 hr. Antiplatelet antibody given 6 hr pre- and 18 hr post-i.v. injection of tumor cells decreased the mean number of CT26 tumor nodules per lung by 57% (range, 47 to 65%) and decreased the mean nodule volume of tumor per lung by 37% (range, 0 to 71%) (124 experimental animals), when compared to the effect of nonimmune serum or irrelevant anti-immunoglobulin antibody in 136 control animals. With Lewis lung carcinoma, antiplatelet antibody decreased the mean number of tumor nodules by 62% (range, 57 to 78%) and decreased the mean nodule volume of tumors by 64% (range, 60 to 77%) using 48 experimental animals and 65 control animals. When tumor cells were given s.c., antiplatelet antibody given 6 hr pre-injection, 18 hr post-injection, and every 48 hr thereafter also decreased the mean number of metastases by 42% in 14 experimental and 15 control animals. With B16 amelanotic melanoma, antiplatelet antibody given 6 hr pre- and 18 hr post-injection decreased the mean number of tumor nodules by 85% and decreased the mean nodule volume of tumors by 66% using 9 experimental and 9 control animals. Similar results were obtained when all three tumors were injected 6 hr after the injection of antiplatelet antibody. However, negative results were obtained if antiplatelet antibody was injected 6 hr after the injection of tumor cells. Since antiplatelet antibody has its maximum effect at 6 hr, it is likely that platelets play their role in the development of pulmonary metastases during the first 12 hr of tumor inoculation.
The combining ability of glycoproteins IIb, IIIa and Ca2+ in EDTA-treated nonaggregable platelets.
Platelets deprived of calcium and incubated at 37 degrees C for 10 min lose their ability to bind fibrinogen or aggregate with ADP when adequate concentrations of calcium are restored. Since the calcium complex of glycoproteins (GP) IIb and IIIa is the presumed receptor for fibrinogen, it seemed appropriate to examine the behavior of these glycoproteins in incubated nonaggregable platelets. No differences were noted in the electrophoretic pattern of nonaggregable EDTA-treated and aggregable control CaEDTA-treated platelets when SDS gels of Triton X-114 fractions were stained with silver. GP IIb and IIIa were extracted from either nonaggregable EDTA-treated platelets or aggregable control platelets with calcium-Tris-Triton buffer and subjected to sucrose density gradient centrifugation or crossed immunoelectrophoresis. With both types of platelets, these glycoproteins formed a complex in the presence of calcium. If the glycoproteins were extracted with EDTA-Tris-Triton buffer, or if Triton-solubilized platelet membranes were incubated with EGTA at 37 degrees C for 30 min, GP IIb and IIIa were unable to form a complex in the presence of calcium. We conclude that inability of extracted GP IIb and IIIa to combine in the presence of calcium is not responsible for the irreversible loss of aggregability that occurs when whole platelets are incubated with EDTA at 37 degrees C.
A new mechanism for tumor induced platelet aggregation. Comparison with mechanisms shared by other tumor with possible pharmacologic strategy toward prevention of metastases.
Because tumor-induced platelet aggregation appears to play a role in the development of certain experimental tumor metastases, we examined the mechanism(s) of tumor-induced platelet aggregation as well as the effect of various anti-platelets agents. Two mechanisms for tumor-induced platelets aggregation have been previously described: (1) a mechanism which requires complement, a stable plasma factor, divalent cation and a sialo-lipo-protein vesicular component of the tumor membrane for platelet aggregation; and (2) a mechanism which operates via the generation of thrombin and requires a phospholipid component of the tumor membrane. We now report a new mechanism of tumor-induced platelet aggregation which is shared by three different tumors: a spontaneously metastatic human melanoma, HM29, a murine melanoma, B16F10, and a carcinogen-induced metastatic murine colon carcinoma, CT26. These tumors do not require cell-surface sialic acid or serum complement as does the first mechanism. They do not require cell-surface phospholipid, as do the tumors representing the other two mechanism. They do not aggregate platelets via the generation of thrombin as do tumors representing the second mechanism. These tumors are unique in that they require a trypsin-sensitive surface protein for activity. The ability of the thrombin-generating tumors to aggregate platelets is uniquely sensitive to two highly specific, synthetic thrombin-competitive inhibitors: DAPA and No. 805. The other two groups of tumors are at least 10 times more sensitive to inhibition of platelet aggregation by elevation of cyclic AMP levels (prostacyclin, 6-keto-PGE1, dibutyryl cyclic AMP) and at least 10 times more sensitive to inhibition of prostaglandin synthesis (indomethacin, ibuprofen). Thus, tumor-induced platelet aggregation is heterogeneous with respect to mechanism of action as well as inhibition by anti-platelet pharmacologic agents. Sensitivity to anti-platelet agents correlates with the mechanism by which tumor cells aggregate platelets.
Reevaluation of the presence of the major antigen Ca++ complex in Bernard-Soulier syndrome platelets. Elastase degradation of the complex in Bernard-Soulier syndrome platelet preparations.
Crossed immunoelectrophoresis of human platelet membranes reveals a major antigen GPIIb-GPIIIa-Ca++ complex which is absent to decreased in Glanzmann's thrombasthenia. In our initial report, we also noted its absence in two patients with Bernard-Soulier syndrome associated with the presence of two neoantigens, and postulated that this may be due to endogenous proteolysis. It now appears that this observation is due to exogenous proteolysis from granulocyte-elastase contamination in the platelet membrane preparation. Purified elastase is capable of degrading the major antigen-Ca++ complex, particularly in its dissociated form, to the pattern initially reported for Bernard-Soulier platelets. Of particular interest is the observation that the elastase-degraded dissociated complex can be reassociated in the presence of Ca++. This indicates that the elastase-sensitive antigenic determinants recognized by the rabbit anti-human platelet membrane antibody are not required for the bonds which reassociate the complex.
A monoclonal anti-platelet antibody with decreased reactivity for autoimmune thrombocytopenic platelets.
Two monoclonal anti-platelet antibodies, 3B2 and 8G11, have been raised that are specific for normal human platelets. 3B2 is unique in that it has decreased reactivity for platelets from 16 patients with autoimmune thrombocytopenic purpura [mean platelet count, 65,000 +/- 6,000 (SEM)]. With 8G11 in an enzyme-linked immunosorbent assay, the mean of the ratios of patient platelet OD to control platelet OD was 0.95 +/- 0.07, whereas with 3B2, the mean of the ratios of patient platelet OD to control OD was 0.24 +/- 0.04, P less than 0.001. With 3B2 the mean of the OD ratios of five patients with autoimmune thrombocytopenic purpura in remission (greater than 150,000 platelets per mm3) compared to controls was 0.80 +/- 0.14. 3B2 did not react with platelets from a patient with Glanzmann's thrombasthenia, in which membranes lack glycoproteins IIb and IIIa (GPIIb and GPIIIa). Platelet membranes were run on crossed immunoelectrophoresis against a rabbit polyclonal anti-human platelet membrane antibody with 125I-labeled purified 3B2 in an intermediate spacer gel. 3B2 reacted with the GPIIb-GPIIIa-Ca2+ complex in the presence of excess Ca2+ and with GPIIb alone in the presence of excess EGTA. When Triton X-100-solubilized platelet membranes were immunoprecipitated with 3B2 plus rabbit anti-mouse IgG, reduced, and run on NaDodSO4/polyacrylamide gel electrophoresis, a single protein band was obtained with a molecular weight of 120,000 (the molecular weight of GPIIb). Thus, the reactivity of monoclonal antibody 3B2 with GPIIb or the GPIIb-GPIIIa-Ca2+ complex appears to be inhibited by the presence of autoantibody on platelets.
Effect of coumadin-induced coagulopoietin plasma on vitamin K-dependent carboxylation of liver microsomes.
Coumadin-treated rabbits have a humoral substance(s) (coagulopoietin) which is capable of elevating vitamin K-dependent coagulation factors when injected into recipient rabbits (Karpatkin & Karpatkin, 1973). Biologic levels of coagulation factors II, V, VII and X; immunologic levels of factors II and X; and vitamin K-dependent liver microsomal carboxylase activity were measured in recipient rabbits receiving coumadin-induced coagulopoietin plasma. Factor II biologic activity increased 3.5-fold compared to the increase in immunologic activity. Factor X biologic activity increased 1.7-fold compared to the increase in immunologic activity. This indicates an increase in specific activity of factors II and X. Coumadin-induced coagulopoietin plasma had no effect on vitamin K-dependent liver microsomal carboxylase activity in vitro. However, livers obtained from recipient animals treated with coumadin-induced coagulopoietin plasma enhanced their carboxylase activity (compared to control animals) 2.4-fold employing endogenous microsomal precursor for carboxylation, and 6.2-fold employing synthetic substrate, phe-leu-glu-glu-val. Thus, coumadin-induced coagulopoietin plasma enhances the biologic activity of vitamin K-dependent coagulation factors II, VII, and X as well as the ex vivo vitamin K-dependent carboxylase activity of liver microsomes.
The spleen and thrombocytopenia.
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Crossed immunoelectrophoresis of human platelet membranes. The major antigen consists of a complex of glycoproteins, GPIIb and GPIIIa, held together by Ca2+ and missing in Glanzmann's thrombasthenia.
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Autoimmune thrombocytopenic purpura in homosexual men.
Since November 1980 we have diagnosed 11 cases of severe autoimmune thrombocytopenic purpura in homosexual men; their mean platelet count (+/- SE) was 16 000 +/- 3000/mm3. All patients have been sexually active with multiple partners and exposed to numerous viruses and drugs. During this period, we also have diagnosed 20 cases of classic autoimmune thrombocytopenic purpura in heterosexual persons, with a normal women to men ratio of 3:1. Eight of nine homosexual patients had elevated platelet IgG compared with normal values in eight of 10 homosexual control subjects having normal hemograms (p less than 0.01). All responded moderately or completely to steroids. The three patients who had splenectomy had excellent responses. Four of five patients had a decreased helper/suppressor T cell ratio compared to healthy controls (p less than 0.001). Circulating immune complexes and total gamma globulin levels were elevated and lymphocytes relatively decreased in homosexual patients compared with homosexual controls (p less than 0.05). Thus, some sexually-active homosexual men seem to have an increased incidence of an immune regulation disorder directed against platelets.
Inhibition of the platelet-aggregating activity of two human adenocarcinomas of the colon and an anaplastic murine tumor with a specific thrombin inhibitor: dansylarginine N-(3-ethyl-1, 5-pentanediyl)amide.
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Abnormal autologous mixed lymphocyte reaction in autoimmune thrombocytopenic purpura.
The autologous mixed lymphocyte reaction (auto-MLR) measures the ability of non-T cells to stimulate autologous T cells to proliferate in tissue culture. The auto-MLR was studied in 11 patients with autoimmune thrombocytopenic purpura (ATP). Seven patients had decreased auto-MLR, which averaged 4440 +/- 3364 cpm (SEM) compared to 15,360 +/- 6905 cpm for simultaneously studied controls. The average of the ratios of cpm incorporated by patients/cpm incorporated by control subjects was 0.20 +/- 0.06 (p less than 0.01). Serum from all 7 patients with low auto-MLR decreased the auto-MLR of normal subjects by an average of 56% +/- 8.5 (p less than 0.001). Preliminary results indicate that the inhibitory effect was mediated by a component of the IgG immunoglobulin fraction of serum. Sera from normal persons and from ATP patients with normal or high auto-MLR did not affect the auto-MLR of normal subjects. It was further shown that non-T cells from 3 of 5 patients with decreased auto-MLR failed to stimulate allogeneic T cells normally. It is concluded that many patients with ATP have decreased auto-MLR apparently due to the presence of a serum blocking factor and, perhaps, a defective stimulatory capacity of non-T-cells.
Platelet aggregating material (PAM) of two virally-transformed tumors: SV3T3 mouse fibroblast and PW20 rat renal sarcoma. Role of cell surface sialylation.
Platelets are required for certain experimental tumor metastases and several lines of tumor cells have been shown to aggregate platelets. We have extracted a sedimentable sialolipoprotein, platelet aggregating material (PAM) from the cell surface of SV40 transformed Balb C3T3 fibroblasts which aggregates heparinized PRP at 2.5 micrograms/ml via the release reaction, following a one minute lag period. A similar extract from non-transformed 3T3 cells has barely measurable activity at 40 micrograms/ml. Gel-filtered platelets (GFP) do not aggregate with PAM. However, PAM aggregation can be restored by addition of 5% plasma but not by fibrinogen. Two plasma components are required: a heat-labile complement component which is activated during the lag period; and a heat-stable factor which is required for platelet aggregation. The pathophysiologic significance of PAM has been examined in ten variant cell lines derived from a spontaneously metastatic renal cell sarcoma of rats, initially induced with polyoma virus (PW20 Wistar-Furth parental lines). These lines were selected in vitro and in vivo from a single line and differed in their capacity to form distant tumors in various organs after subcutaneous injection. These cells were examined for cell surface sialylation, PAM and PAM sialic acid content, since cell surface sialic acid is increased in a variety of tumor tissues and PAM is inhibited by neuraminidase. A good correlation was obtained between in vivo metastatic potential and cell surface sialic acid, r = 0.83, p less than 0.003; cell surface sialic acid and PAM, r = 0.85, p less than 0.002; in vivo metastatic potential and sialic acid content of PAM, r = 0.69, p less than 0.03; and in vivo metastatic potential and PAM, r = 0.68, p less than 0.03. We conclude that platelets may play a role in hematogenous metastasis via the ability of tumor cells to aggregate platelets by cell surface constituents containing sialic acid. The platelet-tumor cell interaction requires activation of the alternate complement pathway and a heat stable plasma factor.
Cumulative experience with a simplified solid-phase radioimmunoassay for the detection of bound antiplatelet IgG, serum auto-, allo-, and drug-dependent antibodies.
A simplified, sensitive, solid-phase radioimmunoassay employing 125I-staphylococcal protein A has been developed that is capable of detecting bound antiplatelet IgG as well as serum auto-, allo-, and drug-dependent antiplatelet antibodies. The simplified assay employs a ratio of test over control platelet counts per minute (cpm) for detection of positive results. All reagents are commercially available. The assay can be performed with as little as 10(6) washed platelets (10 microliters of whole blood) that have been stored for as long as 8 wk at 4 degrees C in microtiter plates. The assay time, employing stored platelets, is 4 hr. Bound platelet IgG is positive in 93% of 46 thrombocytopenic patients with autoimmune disease and correlates inversely with their platelet count, r = -0.65, p less than 0.001. The ability of this assay to detect serum antibody was studied with a rabbit anti-human platelet antibody capable of giving optimal immunoprecipitation with solubilized platelet membranes at a tier of 1:10. The present assay increases the sensitivity of antibody detection 256-fold to a titer of 1:2560. Human serum antiplatelet membrane antibody was positive in 2 of 2 patients with anti-PLA-1 antibody (titers of 1:256 and greater than 1:64); 7 of 12 multiply transfused patients who were refractory to platelet transfusion (2 had titers of greater than 1:256 and greater than 1:32); 5 of 19 patients with autoimmune thrombocytopenic purpura (2 had titers of 1:64 and 1:32); and 10 of 14 patients with clinical histories of drug-dependent antiplatelet antibody (2 had titers of 1:1280 for quinidine and 1:384 for phenazopyridine).