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P A Valant

Publications and source records attributed to P A Valant.

6 recordsLinked to original sources
PubMed1998

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.

Adult↗
PubMed1996

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.

Flow Cytometry↗
PubMed1993

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.

Blood Platelets↗
PubMed1992

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.

Biological Transport, Active↗