Hiroh Yamazaki

Simple method of aequorin loading into platelets using dimethyl sulfoxide.

To investigate changes in Ca2+ concentrations in platelet cytoplasm during the activation, aequorin was loaded into platelets with an incubation of dimethyl sulfoxide (DMSO) in platelet suspension. When washed human platelets (about 5 X 10(9) platelets/microliter) were incubated with 10 microM aequorin and 6% DMSO (final concentrations) for 2 min at room temperature, a part of aequorin penetrated through the platelet membrane into the cytoplasm. The leakage of LDH was very slight indicating membrane damage by DMSO treatment being negligible. The platelet membrane and cytoplasmic components preserved their normal features under electron microscope. Platelet aggregation and ATP release were not affected by the incubation. The amount of permeated aequorin was the largest when DMSO was added stepwisely 6 times for 2 min to reach 6% final concentration. Though about 1/5,000 to 1/10,000 of added aequorin penetrated into platelets, the platelets emitted enough luminescent signals by additions of collagen, thrombin and A 23187. The DMSO method is very simple and can save the incubation time (only 2 min at room temperature) to load aequorin into platelets.

Monoclonal antibody to glycoprotein Ib inhibits both thrombinand ristocetin-induced platelet aggregations

A monoclonal antibody named TM60, which inhibited both thrombin- and ristocetin-induced platelet aggregations, was obtained by hybridoma technique. TM60 inhibited binding of von Willebrand factor to platelets under the presence of ristocetin. The subclass of TM60 was IgG2a. TM60 did not inhibit ADP-, collagen-A-23187-, arachidonic acid- and PAF-induced platelet aggregations, but inhibited polylysine-, polybrene- and cationized ferritin-induced platelet aggregations. ATP-release from platelets induced by thrombin was also inhibited by TM60. Immunoprecipitation and SDS-PAGE experiments demonstrated that TM60 recognized an epitope on GPIb whose molecular weight was 165,000 under non-reduced and 145,000 under reduced conditions.

Consumption of larger platelets with decrease in adenine nucleotide content in thrombosis, disseminated intravascular coagulation, and postoperative state

Abstract Platelet count, volume, aggregation and ATP and ADP contents were measured in 51 healthy volunteers, 27 patients in the acute stage of thrombosis, 29 patients in the recovery stage of thrombosis, 10 patients with typical DIC syndrome during the course of cancer, and 39 patients one day before, and one day and one week after abdominal operation. A positive correlation was found between platelet mode volume and agaregability in the healthy subjects. In DIC, acute stage of thrombosis, and one week after abdominal operation patients, platelet volume and ADP content decreased significantly. Platelet aggregation increased in thrombosis and in the postoperative state patients, while it decreased in the DIC subjects. In DIC, a decrease in ATP and ADP contents was marked as compared with the other groups. A positive correlation was observed between platelet volume and the adenine nucleotide content. The results suggest that larger platelets with hyperaggregability were consumed easily in thrombosis, DIC and hemostatic plug formation after operation. A mechanism to induce platelet release reaction following secondary aggregation in vivo may be present in thrombosis and postoperative state.

Autoantibody against platelet glycoprotein II b/ III a in a patient with non-Hodgkin's lymphoma

An autoantibody to platelet glycoprotein (GP) II b/III a was produced in a 38 year-old woman who had a previous history of the malignant lymphoma of the stomach. The aggregations of the patients platelets showed losses of the primary waves in response to ADP and epinephrine and marked hypoaggregation in response to collagen, while agglutination by ristocetin was normal. Crossed immuno-electrophoresis (CIE) of her platelets solubilized by 1% Triton X-100 revealed an abnormal biphasic precipitate line of GP II b/III a complex. Nine months later, she developed severe thrombocytopenia along with a relapse of the lymphoma in the cervical lymph nodes. The patients IgG, which was collected during her thrombocytopenic period and purified, inhibited ADP-, epinephrine- and collagen-induced aggregations of normal platelets. In CIE, the 125I-labelled IgG of the patient, inserted into the intermediate gel, was incorporated into the precipitation line of the GP II b/III a complex of normal platelets. Radiation treatment to the cervical lymph nodes dramatically normalized both the function and the count of the patients platelets. From these findings, it is suggested that an autoantibody to the GP II b and/or III a was produced by the lymphoma cells.

Localization of adhesive proteins in two newly subdivided zones in electron-lucent matrix of human platelet α-granules

SummaryPlatelet α-granules have been reported to consist of two zones, nucleoid and electron-lucent matrix, with different densities under electron microscopy. When washed human platelets were prepared by a rapid freeze-substitution method using liquid helium, we found that the electron-lucent matrix could be further subclassified into two zones having different densities: the intermediate and the light zones. The light zone was located at the periphery opposite the most dense nucleoid and contained several tubular structures with diameters of about 20 nm. The intermediate zone often laid between the nucleoid and light zone. By careful inspection, intermediate and light zones could even be identified in the platelets embedded in Lowicryl K4M, which where then used to localize several adhesive proteins in these two zone by immunocytochemical studies using the respective polyclonal antibodies. Fibrinogen, thrombospondin, and fibronectin were detected only in the intermediate zone. In contrast, von Willebrant factor (vWF) was localized only in the light zone, suggesting an association between vWF and the tubular structures in the light zone. In the nucleoid, none of these adhesive proteins were detected. Glycoprotein IIb/IIIa, a receptor for these adhesive proteins on the platelet surface, was detected not only on the outer surface of the cell membranes but also on the inner surface of the α-granule membrane. These data indicate that two zones with different densities in electron-lucent matrix and functions exist in the platelet α-granules.

Enhancement by IL-1β and IFN-γ of platelet activation: Adhesion to leukocytes via GMP-140 / padgem protein (CD62)

Abstract We have examined the effect of inflammatory cytokines on the platelet activation. IL-1β and IFN-γ were found to enhance the adhesion of thrombin-treated platelets to monocytic leukemia cells (U937), when the adhesion was assayed by platelet-mediated cell agglutination. The agglutination was inhibited by a monoclonal anti-GMP140 antibody or EDTA, suggesting that the enhanced platelet adhesion to the leukemic cells was mediated by GMP140. In addition, these cytokines also increased the release of 5-HT from platelets in the presence of a low concentration of thrombin. These data suggest that platelet functions are regulated by the cytokines and that activated platelets participate in inflammatory process.

Cleavage site of calcium-dependent protease in human platelet membrane glycoprotein ib

Chicken muscle-derived m-type calcium-dependent protease cleaved purified glycoprotein Ib alpha-chain (GPIb alpha, Mr 130,000) from human platelets into two fragments (Mr 100,000 and Mr 38,000) in the presence of 5 mM calcium. With partially purified glycoprotein Ib (alpha beta-dimer), an appearance of a fragment of Mr 100,000 was also demonstrated after treatment with both the m-type and human platelet-derived mu-type protease. These processes in glycoprotein Ib were inhibited by inhibitors of calcium-dependent proteases, 50 muM E-64-C or 0.2 mM leupeptin and by the chelation of calcium. Using two-dimensional gel electrophoresis system, release of glycocalicin in addition to 100 kDa fragment was demonstrated by calcium-dependent proteases. Then surface-labeled platelets were stimulated with A23187 in the presence of 5mM calcium. Under this condition, endogenous calcium-dependent protease is activated. Of the labeled glycoproteins, glycocalicin and glycoprotein V but not 100 kDa fragment were released from the platelet membrane. The released glycocalicin was further digested into a fragment of Mr 100,000 by the addition of m-type calcium-dependent protease. These results showed (i) that GPIb alpha was hydrolyzed by exogenous calcium-dependent proteases in two points and glycocalicin and 100 kDa fragment were produced and (ii) that endogenous protease cleaved GPIb alpha at one point and released glycocalicin.

Immunocytochemical evidence for the translocation of α-granule membrane glycoprotein IIb/IIIa (integrin αIIbβ3) of human platelets to the surface membrane during the release reaction

SummaryThe localization of glycoprotein (GP) IIb/IIIa (integrin αIIbβ3) in both resting and thrombin-activated platelets was studied immunocytochemically. By the pre-embedding method where only the GP IIb/IIIa molecules on the surface of platelets were immunostained, the distribution of protein A-colloidal gold label was randomly distributed along the surface membrane of resting platelets at a density of 18.0±2.7 gold particles/μm of membrane. At 15 s after stimulation by 0.1 U/ml of thrombin in an unstirred platelet suspension, the spheroid-shaped platelets with pseudopodia still had normal numbers of α-granules, and the density of gold particles was 19.7±3.6 particles/μm. At 5 min, the α-granules were no longer present because of the release reaction, and the density of gold particles significantly increased (27.0±3.7 particles/μm; p<0.01). In immunostained ultra-thin frozen sections, the gold particles were detected not only on the surface membrane, including the open canalicular system (OCS), but also on the α-granule membranes of resting platelets. At 30 s after thrombin stimulation the α-granules fused with the OCS, resulting in the formation of a swollen OCS, which still had gold particles on its membrane. At 5 min, the gold particles were detected on the membrane of the swollen OCS located near the surface membrane, while very few gold particles were present on the membrane of the OCS in the central part of the platelets. These results demonstrate that α-granule membrane GPIIb/IIIa translocates to the surface membrane through the membrane of the OCS. Also the translocation of α-granule membrane GPIIb/IIIa gives rise to an actual increase in GPIIb/IIIa on the surface membrane during the release reaction induced by thrombin.

Glycoprotein Ib distribution on the surface of platelets in resting and activation states: An electron microscope study

SummaryUsing a monoclonal antibody (TM60) against glycoprotein (GP) Ib, we determined immunocytochemically how GPIb is distributed on the platelet surface. When glutaraldehyde-fixed platelets were incubated with TM60, a uniform distribution of ferritin particles which represent the localization of GPIb was observed on the surface membrane of platelets. The particles were distributed at intervals of about 100 nm. The number of ferritin particles on the surface of one side were 2070–4150 (2940 ± 790; mean ±s.d.,n = 10) under the scanning electron microscope. The distribution of ferritin particles was somewhat disarranged on the surface of unfixed platelets incubated with TM60 compared to that in the fixed platelets. Cluster-like structures of ferritin particles were observed in several places. When platelets were activated with ristocetin or thrombin, the distribution of ferritin particles was disturbed and cluster formation was observed in several places on the surface. These findings suggest that GPIb is uniformly distributed on the surface of platelets in the resting state, and that cluster formation occurs during activation of platelets.

A new variant of thrombasthenia with abnormally glycosylated GP IIb/IIIa

A 15 year-aged Japanese girl with a life long mild purpura was found as a variant type of thrombasthenia. Basic tests revealed prolonged bleeding time, border-line clot retraction, no coagulation defect, no giant platelets and mild thrombocytopenia (70,000-110,000/microliters). Neither ADP, epinephrine nor collagen aggregated her platelet rich plasma. Thrombin (0.1U/ml) caused slightly decreased aggregation of her washed platelets and about 20% normal production of thromboxane B2. PAS-stained SDS-PAGE of her whole platelets showed markedly decreased GP IIb and IIIa. However, crossed immunoelectrophoresis (CIE) against anti-whole platelets antibody showed a normal amount of GP IIb/IIIa complex in her whole platelets solubilized with 1% Triton X-100. CIE using monospecific anti-GP IIb/IIIa complex antibody showed normal dissociation of the patients GP IIb/IIIa complex into two new bands in the presence of EDTA. Crossed affino-immunoelectrophoresis with the first dimension containing Concanavalin A revealed that the patients GP IIb/IIIa was much less shifted to the cathode than controls. Immunoprecipitation lines of her GP IIb/IIIa complex were excised from unstained CIE using anti-GP IIb/IIIa antibody and subjected to the silver-stained reduced SDS-PAGE, which showed two protein bands with molecular weights of 125KD and 108KD, corresponding to GP IIb alpha and GP IIIa, respectively. These results suggest that the platelets of this apparently thrombasthenic patient have an antigenically normal but abnormally glycosylated GP IIb/IIIa complex, which is functionally abnormal because of abnormal glycosylation.