A flow cytometric assay of platelet activation marker P-selectin (CD62P) distinguishes heparin-induced thrombocytopenia (HIT) from HIT with thrombosis (HITT).

Heparin induced thrombocytopenia (HIT) is a well-known complication of heparin administration but usually resolves upon discontinuation without sequelae. However, a small proportion of HIT patients develop thrombosis associated with HIT, designated as HITT, which is often life-threatening and may lead to gangrene and amputations. Existing laboratory methods of confirming HIT/HITT do not distinguish between HIT and HITT. We report a flow cytometric assay of platelet activation marker CD62P to distinguish the effects of addition of HIT vs. HITT plasma to normal blood. Briefly, normal whole blood was incubated with platelet-poor plasma from 12 patients with HITT, 30 with HIT, and 65 controls, in presence and absence of heparin, and expression of CD62P was assayed by flow cytometry. When the ratios of fluorescent intensity of CD62P with heparin divided by that without heparin were compared, HITT plasma induced significantly higher ratios than HIT plasma (HITT ratios approximately 2.5 vs. HIT ratios approximately 1.2; p <0.001). Eleven of 12 HITT patients were positive by this test but only 5 of 30 HIT patients were positive (p <0.0005). In a case of HIT with silent thrombosis, this assay gave a positive results prior to clinically evident thrombosis. In conclusion, this method distinguishes HITT from HIT and may be clinically useful in the detection of HITT, allowing early intervention for preventing catastrophic thrombosis.

Anti-CD36 autoantibodies in thrombotic thrombocytopenic purpura and other thrombotic disorders: identification of an 85 kD form of CD36 as a target antigen.

The presence of anti-CD36 antibodies in plasma of patients with thrombotic thrombocytopenic purpura (TTP), idiopathic thrombocytopenic purpura (ITP), and heparin-induced thrombocytopenia without/with thrombosis (HIT/HITT) has been examined by immunoblots, and a monoclonal antibody capture assay, the platelet-associated IgG characterization assay (PAICA). Results with PAICA showed that 73% (8/11) of patients with TTP were positive, and 71% (10/14) by immunoblots. With ITP, 20% (6/30) were positive by PAICA and 19% (3/16) by immunoblots; HIT, 30% (3/10) were positive by PAICA and 60% (6/10) by immunoblot; HITT, 50% (2/4) by PAICA and 100% (4/4) by immunoblot. Purification of CD36 by fast protein liquid chromatography (FPLC) from Triton X-100 extracts of normal platelet membranes resulted in the isolation of two different forms: the classic 88 kD form, and a second, lighter 85 kD form. Our data indicated that the patients' plasma autoantibodies reacted strongly with the 85 kD form. Conventional monoclonal and polyclonal antisera produced to the 88 kD form reacted strongly with the 88 kD form but weakly with the 85 kD form. These results confirm the possible importance of anti-CD36 antibodies in the pathophysiology of TTP and other thrombocytopenias and demonstrate the presence of a previously unrecognized target antigen for these antibodies.

Thrombotic thrombocytopenic purpura plasma enhances platelet-leucocyte interaction in vitro.

In thrombotic thrombocytopenic purpura (TTP), intravascular platelet aggregation and formation of platelet-rich thrombi impair the microcirculation. TTP plasma has been shown to induce aggregation of normal platelets in vitro. The present study investigates the formation of activated platelet aggregates (aPAg) induced by TTP plasma, with particular attention to their binding to leucocytes (LPAg). Results were compared with the effects of plasmas from normal controls (CTL) and from patients with immune thrombocytopenic purpura (ITP) or thrombosis (THR). Following addition of test plasma to normal whole blood (WB), aPAg and LPAg were assayed by flow cytometry using mAbs against CD41 (platelet marker), CD62p (platelet activation marker) and CD45 (pan-leucocyte marker), Compared to control plasma, TTP plasma was more potent than ITP or THR plasma in increasing aPAg: only TTP plasma significantly promoted leucocyte binding to give increased LPAg. Prior removal of neutrophils (PMN) from WB by beads coated with anti-CD15 mAb largely prevented formation of aPAg and LPAg. However, TTP plasma added to normal platelet-rich plasma significantly increased aPAg, which suggested possible hindrance of aPAg formation by erythrocytes and other leucocytes in PMN-depleted blood. We concluded that TTP plasma was most potent in the induction of aPAg and unique in promoting LPAg formation in WB. Neutrophils, and not other leucocytes, appear to be essential for LPAg formation. Enhanced PMN-platelet interaction in the microcirculation may facilitate platelet adhesion to vessel walls and promote the formation of platelet-rich microthrombi in TTP.

Activated platelet aggregates in thrombotic thromboctyopenic purpura: decrease with plasma infusions and normalization in remission.

Circulating activated platelet aggregates (aPA) were assayed by flow cytometry employing mAb alpha-CD62p in eight patients with thrombotic thrombocytopenic purpura (TTP). Elevation of aPA was observed in all patients in active stages of TTP; aPA normalized in remission. Plasma infusions with plasmapheresis decreased aPA in responding patients. The rise and fall of aPA preceded relapses and improvements, respectively. These changes were seen prior to the traditional indicators, LDH, haematocrit, and platelet count. Incubation of plasma from TTP patients with normal whole blood induced formation of aPA; this effect was significantly greater than that of plasmas from ITP patient controls (P < 0.01), suggesting the presence of an aPA-promoting factor in TTP plasma. Parallel experiments using a platelet aggregometer failed to detect effect of TTP plasma on normal blood. In summary, aPA appear to be a marker of disease activity, rising with relapse, falling with plasma therapy, and normalizing in remission. The flow cytometric assay of aPA is more sensitive than aggregometry in detecting the putative aPA-promoting factor in TTP.

Na(+)-independent Active H(+) Extrusion and HCO(3)(-)/C1(-) Exchange Contribute to Cytoplasmic pH Regulation in the Human Platelet.

Homeostatic regulation of cytoplasmic pH (pH(eyt)) against acid and alkaline challenges was studied in the human platelet using the intracellular indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Activation of a Na(+)/H(+) exchanger in the plasma membrane is a known mechanism by which the platelet resists cytoplasmic acidification. The present study demonstrates an additional Na(+)-independent H extrusion mechanism which persists at low external Na(+) concentration in the presence of 100 µM ouabain. This mechanism is inhibited by rotenone, oligomycin and an inhibitor of glycolysis, and is tentatively identified as a plasmalemmal H(+)-ATPase. The Na(+)-independent H extrusion mechanism partially restores cytoplasmic pH (pH(eyt)) after the cytoplasm is acidified by addition of 1 µM nigericin. The restoration process is energy-dependent and has a t(1/2) of 7-21 min. The Na(+)-independent H(+) extrusion mechanism is also shown capable of maintaining pH(eyt) ≊ 6.0 against an acidic pH, of 5.3 in an energy-dependent manner. The present study also revealed a Na(+)-independent, DIDS-inhibitable Cl(-)/HCO(3)(-) exchange activity. It removes alkaline equivalents from the cytoplasm with a half-time of 2.0 ± 0.4 min after alkaline loading with 25 mM NH(4)Cl. Its activity was also revealed in chloride to gluconate and gluconate to chloride perturbations of the external medium which raised or lowered pH(eyt) by 0.17 ± 0.05 or 0.14 ± 0.04 units, respectively. The activity of the anion exchanger allows the platelet to maintain pH(ext) = 7.00 ± 0.11 at the alkaline pH(ext) of 8.25. The combined activities of the Na(+)-independent H(+) extrusion and Cl(-)/HCO(3)(-) exchange mechanisms make the platelet cytoplasm very resistant to changes in external pH. For variation of pH(ext) between 5.0 and 8.5, pH(eyt) varies between 6.0 and 7.0, or roughly one-third as much.

The Ca(2+)-extruding ATPase of the human platelet creates and responds to cytoplasmic pH changes, consistent with a 2 Ca2+/nH+ exchange mechanism.

The Ca(2+)-extruding ATPase pump of the human platelet was studied in situ by measuring Ca2+ extrusion from quin2-overloaded platelets (Johansson, J.S., Haynes, D.H. 1988. J. Membrane Biol. 104:147-163). Cytoplasmic pH (pHcyt) was measured by BCECF fluorescence in parallel experiments. The pump was studied by raising the cytoplasmic free Ca2+ to 2.5 microM and monitoring active Ca2+ extrusion into a Ca(2+)-free medium. The pump was shown to perturb pHcyt, to not respond to changes in membrane potential and to respond to imposed changes in pHcyt in a manner consistent with the Ca2+ pump acting as a 2 Ca2+/nH+ exchanger. (i) Raising the external pH (pHext) from 7.40 to 7.60 lowers the Vmax of the pump in basal condition (Vmax,1) from 110 +/- 18 to 73 +/- 12 microM/min (= mumol/liter cell volume/min). (ii) Lowering pHext to 7.13 raised Vmax,1 to 150 +/- 15 microM/min. (iii) In an N-methyl-D-glucamine (NMDG+) medium, the pump operation against high [Ca2+]cyt acidifies the cytoplasm by -0.36 +/- 0.10 pH units, and the pump becomes self-inhibited. (iv) Use of nigericin to drive pHcyt down to 6.23 reduces the Vmax,1 to 18 +/- 11 microM/min. (v) Alkalinization of the cytoplasm by monensin in the presence of Na+ raises the Vmax,1 (basal state with Km,1 = 80 nM) to 136 +/- 24 microM/min, but also activates the pump fourfold (Vmax,2 = 280 +/- 28 microM/min; Km,2 = 502 +/- 36 nM). (vi) Transient elevation of pHcyt by NH4Cl at high [Ca2+]cyt activates the pump eightfold (Vmax,2 > or = 671 +/- 350 microM/min). The large activation by alkaline pHcyt at high [Ca2+]cyt can be explained by Ca(2+)-calmodulin activation of the pump (Valant, P.A., Adjei, P.N., Haynes, D.H. 1992. J. Membrane Biol. 130:63-82) and by increased Ca2+ affinity of calmodulin at high pH.

Rapid Ca2+ extrusion via the Na+/Ca2+ exchanger of the human platelet.

This communication reports the kinetics of the Na+/Ca2+ exchanger and of the plasma membrane (PM) Ca2+ pump of the intact human platelet. The kinetic properties of these two systems were deduced by studying the rate of Ca2+ extrusion and its Na+ dependence for concentrations of cytoplasmic free Ca2+ ([Ca2+]cyt) in the 1-10-microM range. The PM Ca(2+)-ATPase was previously characterized (Johansson, J.S. Haynes, D.H. 1988. J. Membrane Biol. 104:147-163) for [Ca2+]cyt < or = 1.5 microM with the fluorescent Ca2+ indicator quin2 (Kd = 115 nM). That study determined that the PM Ca2+ pump in the basal state has a Vmax = 0.098 mM/min, a Km = 80 nM and a Hill coefficient = 1.7. The present study extends the measurable range of [Ca2+]cyt with the intracellular Ca2+ probe, rhod2 (Kd = 500 nM), which has almost a fivefold lower affinity for Ca2+. An Appendix also describes the Mg2+ and pH dependence of the Kd and fluorescence characteristics of the commercially available dye, which is a mixture of two molecules. Rates of active Ca2+ extrusion were determined by two independent methods which gave good agreement: (i) by measuring Ca2+ extrusion into a Ca(2+)-free medium (above citation) or (ii) by the newly developed "ionomycin short-circuit" method, which determines the ionomycin concentration necessary to short circuit the PM Ca2+ extrusion systems. Absolute rates of extrusion were determined by knowledge of how many Ca2+ ions are moved by ionomycin per minute. The major findings are as follows: (i) The exchanger is saturable with respect to Ca2+ with a Km = 0.97 +/- 0.31 microM and Vmax = 1.0 +/- 0.6 mM/min. (ii) At high [Ca2+]cyt, the exchanger works at a rate 10 times as large as the basal Vmax of the PM Ca2+ extrusion pump. (iii) The exchanger can work in reverse after Na+ loading of the cytoplasm by monensin. (iv) The PM Ca2+ extrusion pump is activated by exposure to [Ca2+]cyt > or = 1.5 microM for 20-50 sec. Activation raises the pump Vmax to 1.6 +/- 0.6 mM/min and the Km to 0.55 +/- 0.24 microM. (v) The Ca2+ buffering capacity of the cytoplasm is 3.6 mM in the 0.1 to 3 microM range of [Ca2+]cyt. In summary, the results show that the human platelet can extrude Ca2+ very rapidly at high [Ca2+]cyt. Both the Na+/Ca2+ exchanger and Ca2+ pump activation may prevent inappropriate platelet activation by marginal stimuli.

Calibration methods and avoidance of errors in measurement of intracellular pH (pHcyt) using the indicator bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) in human platelets.

Determinations of pHcyt in suspensions of human platelets using BCECF [bis(carboxyethyl)-5(6)-carboxyfluorescein] can be seriously biased by leakage of the fluorescent indicator. Two methods ("pH jump" and "Mn(2+)") are presented for determining the fraction of external indicator (B ext) and eliminating this error. Both methods rely on rapid perturbations (pH jump or Mn(2+) addition), which affect the fluorescence of the external dye immediately and the intracellular dye more slowly. Identical values ofB ext are reported. Failure to correct for dye leakage can result in overestimation of pHcyt by as much as 0.4 unit at physiological external pH (pHext). Two methods of calibration of the cytoplasmic signal were compared after correcting forB ext: the "digitonin lysis" method and the "nigericin calibration" method. In the digitonin method the dye is released at the end of the experiment and the dependence of its fluorescence is determined as a function of pH. The method assumes that the fluorescence and titration characteristics of the dye in the cytoplasm are not different from those in solution. It gives pHcyt=6.75±0.07 for pHext=7.3. In the nigericin method, 150 mM external K(+) and 10 µM nigericin are used for the purpose of setting pHcyt=pHext to accomplish anin situ calibration. The method was complicated by extra leakage induced by nigericin. Assuming that the ionophore could equilibrate pH in the alkaline range, the fluorescence of the anionic form of BCECF in the cytoplasm would be 15% lower than in solution and pHcyt would be 0.3 unit higher than presented above. A number of observations favor the digitonin lysis method of calibration. The fluorescence polarization of BCECF in platelets is small and indistinguishable from that in solution (0.000±0.022). The spectrofluorimetric characteristics of the intracellular dye are identical to those in solution (150 mM NaCl or KCl). There was no evidence for self-quenching or binding to cellular elements for cytoplasmic BCECF concentrations up to 1.8 mM. The following agents are capable of introducing error: (1) the Na(+) substituteN-methyl-D-glucamine doubles theK d and decreases by 13% the ΔF max of BCECF; (2) the Na(+)/H(+) exchange inhibitor amiloride quenches BCECF fluorescence and is intrinsically fluorescent; and (3) bovine serum albumin (used to remove nigericin) quenches external BCECF with kinetics mimicking acidification of the cytoplasm.