Hemodynamic effects of eating and prolonged supine position in healthy subjects studied under clinical-pharmacological test conditions.

The influences of both being in a supine position for a prolonged period and food intake on cardiovascular variables were studied under clinical-pharmacological test conditions. In a randomized crossover design study without drug or placebo administration, 6 healthy male volunteers received a light standard meal before and during test A and fasted in test B. In both tests, while they were continuously supine for more than 8 h, a synchronous recording of cardiovascular variables was done at 24, 26 and 28 min after starting the supine position (first recordings) and 25 times from 2 to 480 min after the first recordings. Using a multifactorial statistical analysis, each parameter was evaluated regarding the factors eating and time of supine recording. Eating led to a significant decrease in diastolic and mean blood pressure, PQ time and QS2 time, a downward trend in systemic vascular resistance and an upward trend in systolic blood pressure and cardiac output. When the subjects remained in a supine position for prolonged periods, significant increases in systolic, diastolic, mean blood pressure and systemic vascular resistance were noted as well as significant decreases in cardiac output and QS2 time. Thus, eating and remaining in a supine position for prolonged periods should be considered as sources of bias in clinical-pharmacological studies on cardiovascular drug effects and accompanying placebo controls.

Medication adherence, self-care behaviour and knowledge on heart failure in urban South Africa: the Heart of Soweto study.

BACKGROUND: There is a paucity of data on treatment adherence in patients with chronic heart failure (CHF) in Africa. METHODS: We examined the pattern of treatment adherence, self-care behaviour and treatment knowledge in 200 consecutive patients with CHF attending the Chris Hani Baragwanath Hospital, Soweto, South Africa via a combination of questionnaire (100%, n = 200) and pill count (41%, n = 82). RESULTS: Mean age was 56 +/- 14 years, 157 were black African (79%) and 109 (55%) were male. CHF-specific treatment included loop diuretics (93%), beta-blockers (84%), ACE inhibitors (74%), spironolactone (64%) and cardiac glycosides (24%); mean number of medications was 6 +/- 2. Overall, 71% (58 of 82) adhered to their prescribed CHF regimen and individual medication adherence ranged from 64 to 79%. Behavioural adherence varied from 2.5 to 98%. Patient treatment knowledge was poor; 56% could not name medication effects or side effects. However, an average knowledge score of 69% was achieved on 10 questions concerning CHF management. CONCLUSION: As in other regions of the world, non-adherence to complex CHF treatment is a substantial problem in Soweto. Our data confirm the need for a dedicated CHF management programme to optimise CHF-related outcomes in a low-resource environment.

Effect of locally applied trapidil on norepinephrine- and dinoprost-evoked hand vein constriction in healthy men.

OBJECTIVE: In this study the effect of locally administered trapidil on human hand veins was examined. SUBJECTS: 10 healthy male volunteers aged 20 - 30 years were included. METHOD: The dorsal hand vein compliance technique was used. In a crossover design the influence of locally infused trapidil (mainly 5 - 400 microg/min) on hand veins preconstricted with either norepinephrine (adrenoceptor agonist) or dinoprost (prostaglandin F2alpha) was investigated. Preconstriction reduced the vein diameter by about 80% with continuous local infusion of individually determined doses of norepinephrine in the range 11 - 1,000 ng/min and dinoprost in the range 90 - 5,600 ng/min. Blood pressure, cardiac function (electrocardiogram) and skin temperature of the hand infused were monitored. RESULTS: Locally applied trapidil produced a dose-dependent dilation of hand veins preconstricted with norepinephrine and dinoprost. The corresponding ED50 values of trapidil did not differ significantly on an intraindividual comparison. Clinically important side effects with the drugs used were not observed. CONCLUSIONS: The results indicate that trapidil has a direct dilating action on superficial veins in humans. This effect is apparently achieved without involvement of adrenoceptors or prostanoid receptors in venous smooth muscle.

[Differential influence of immune therapy on relapses and progression in multiple sclerosis: interpretation and therapeutic consequences]. / Unterschiedliche Wirkung der Immuntherapie auf Schübe und schleichende Progression bei Multipler Sklerose: Deutung und Konsequenzen für die Therapie.

It is well established that relapses can be suppressed by different substances in patients with relapsing-remitting multiple sclerosis (MS). In contrast, patients with progressive forms of MS do hardly respond to immune therapy. Therefore, start of immune therapy after the first relapse has been proposed, especially in order to prevent degeneration and disability. This view is challenged in the present review. Actually no evidence exists in support of a retardation or an attenuation of secondary progression by early immune therapy. Widespread degeneration occurs early and progresses independently from inflammatory plaques. Therefore, autoimmunity per se is no adequate paradigm to explain MS-pathogenesis entirely. A virus/superantigen-dualism is proposed to explain the different parts of MS, instead. It is concluded that evidence-based immune therapy should be adapted to the actual inflammatory activity of the disease. A suitable parameter for this purpose is the interval between 2 relapses.

The inflammatory action of CD40 ligand (CD154) expressed on activated human platelets is temporally limited by coexpressed CD40.

Recently, we have demonstrated that human platelets carry preformed CD40 ligand (CD154) molecules, which rapidly appear on the platelet surface following stimulation by thrombin. Once on the surface, platelet CD154 induces an inflammatory reaction of CD40-bearing endothelial cells. This study shows that strong platelet agonists other than thrombin also lead to the expression of CD154 on the platelet surface. At the same time, several lines of evidence are presented that together indicate that thrombotic events in the vasculature are generally accompanied by activation of the inflammatory potential of platelet CD154. This study also reports the constitutive expression of CD40, the receptor for CD154, on platelets. The binding of CD154 to coexpressed CD40 in the platelet aggregate leads within minutes to hours to the cleavage of membrane-bound surface CD154 and the release of an 18-kd soluble form of the molecule. Soluble CD154 (sCD154), in contrast to transmembrane CD154, can no longer induce an inflammatory reaction of endothelial cells. These findings indicate that the interaction of platelet CD154 with CD40 on neighboring cells is temporally limited to prevent an uncontrolled inflammation at the site of thrombus formation. Thus, similar to the very tight regulation of the CD154-CD40 interaction in the immune system, an effective mechanism controls the inflammatory potential of platelet CD154 in the vascular system. (Blood. 2001;98:1047-1054)

Serum ras (p21) as a marker for occupationally derived lung cancer?

Certain subsets of the population are especially sensitive to carcinogens, and this can be determined using molecular biological methods. In the literature there has been evidence presented for the use of p21ras (ras) as a tumor marker for human carcinogenic substances such as asbestos, polycyclic aromatic hydrocarbons, and vinyl chloride in the workplace. In this study we have examined whether serum ras could serve as a biomarker for the early detection of occupationally derived lung cancer, with an emphasis on Schneeberger (radon-induced) lung cancer. Sera were taken from 65 male tumor patients. Fifty-nine patients suffered from primary lung cancer (including 18 patients with Schneeberger lung cancer and 12 patients with asbestos-related lung cancer). Additionally, 29 patients with non-malignant lung disease, and a healthy control group (44) including 32 former uranium miners of SDAG Wismut exposed to ionizing radiation (radon and its decay products) were examined. Ras protein was determined via three different methods: 1) immunoprecipitation followed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting; 2) SDS-PAGE using 5-17% gradient gels followed by Western blotting; 3) pre-incubation with Blue Sepharose, SDS-PAGE on 5-17% gradient gels, and Western blotting. The results show that 1 ng ras protein was measurable in serum standards. This protein could not be detected in patient sera or in sera from any of the study groups. Thus, ras cannot be considered useful as a marker for the early detection of asbestos-induced or Schneeberger lung cancer.

Protein-tyrosine-phosphatase-mediated epidermal growth factor (EGF) receptor transinactivation and EGF receptor-independent stimulation of mitogen-activated protein kinase by bradykinin in A431 cells.

Transactivation of the epidermal growth factor (EGF) receptor (EGFR) has been proposed to represent an essential link between G-protein-coupled receptors and the mitogen-activated protein kinase (MAPK) pathway in various cell types. In the present work we report, in contrast, that in A431 cells bradykinin transinactivates the EGFR and stimulates MAPK activity independently of EGFR tyrosine phosphorylation. Both effects of bradykinin are mediated by a pertussis-toxin-insensitive G-protein. Three lines of evidence suggest the activation of a protein tyrosine phosphatase (PTP) by bradykinin: (i) treatment of A431 cells with bradykinin decreases both basal and EGF-induced EGFR tyrosine phosphorylation, (ii) this effect of bradykinin can be blocked by two different PTP inhibitors, and (iii) bradykinin significantly increased the PTP activity in total A431 cell lysates when measured in vitro. The transmembrane receptor PTP sigma was identified as a putative mediator of bradykinin-induced downregulation of EGFR autophosphorylation. Activation of MAPK in response to bradykinin was insensitive towards AG 1478, a specific inhibitor of EGFR tyrosine kinase, but was blocked by wortmannin or bisindolylmaleimide, inhibitors of phosphatidylinositol 3-kinase (PI3-K) and protein kinase C (PKC) respectively. These results also suggest that the bradykinin-induced activation of MAPK is independent of EGFR and indicate a pathway involving PI3-K and PKC. In addition, bradykinin evokes a rapid and transient increase in Src kinase activity. Although Src does not participate in bradykinin-induced stimulation of PTP activity, inhibition of Src by 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine leads to an increase in MAPK activation by bradykinin. Our results suggest that in A431 cells the G(q/11)-protein-coupled bradykinin B(2) receptor may stimulate PTP activity and thereby transinactivate the EGFR, and may simultaneously activate MAPK by an alternative signalling pathway which can bypass EGFR.

Biomarkers of genetic damage and inflammation in blood and bronchoalveolar lavage fluid among former German uranium miners: a pilot study.

Former East German uranium miners who are known to have been exposed to radon are estimated to be at high risk for lung carcinogenesis. Among these miners over 200 occupationally caused lung cancer cases are expected to occur each year, resulting in a total of 7,000-24,000 excess lung cancer cases in the coming years. It is still unknown whether there is a correlation between biomarkers and the exposure of the uranium miners to ionizing radiation that might enable us to trace those miners with high lung cancer risk. The primary aim of this pilot study was to test the possibility of performing a biomarker study in this unique cohort of former uranium miners in spite of several limitations that had to be taken into consideration when comparing them with healthy controls, such as old age, age-dependent diseases and potential confounding artefacts from dissimilar smoking patterns. The second aim was to test a range of biomarkers for DNA damage and inflammation in leukocytes and bronchoalveolar fluid for their ability to detect biological effects. In this cohort of miners we found an increased frequency of chromosomal aberrations in blood lymphocytes and an increased prevalence of both fibronectin and tumour necrosis factor alpha in the bronchoalveolar fluid.

A key role of adenosine diphosphate in the irreversible platelet aggregation induced by the PAR1-activating peptide through the late activation of phosphoinositide 3-kinase.

Although adenosine diphosphate (ADP), per se, is a weak platelet agonist, its role as a crucial cofactor in human blood platelet functions has now been clearly demonstrated in vitro and in vivo. The molecular basis of the ADP-induced platelet activation is starting to be understood since the discovery that 2 separate P2 purinergic receptors may be involved simultaneously in the activation process. However, little is known about how ADP plays its role as a cofactor in platelet activation and which signaling pathway initiated by a specific agonist can be modulated by the released ADP. To investigate these points, we took advantage of a model of platelet activation through the thrombin receptor PAR1 in which both ADP scavengers and phosphoinositide 3-kinase (PI 3-kinase) inhibitors have been shown to transform the classical irreversible aggregation into a reversible one. We have observed that, among the different PI 3-kinase products, the accumulation of phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P(2)] was dramatically and specifically attenuated when ADP was removed by apyrase treatment. A comparison between the effects of PI 3-kinase inhibitors and apyrase strongly suggest that the late, ADP-dependent, PtdIns(3,4)P(2) accumulation is necessary for PAR1-induced irreversible aggregation. Using selective antagonists, we found that the effect of ADP was due to the ADP receptor coupled to inhibition of adenylyl cyclase. Finally, we found that both ADP and PI 3-kinase play an important role in PAR1-dependent reorganization of the cytoskeleton through a control of myosin heavy chain translocation and the stable association of signaling complexes with the actin cytoskeleton.

Serum levels of pantropic p53 protein and EGF-receptor, and detection of anti-p53 antibodies in former uranium miners (SDAG Wismut).

BACKGROUND: The oncogene product EGF-receptor (EGF-R), the tumor suppressor gene product p53, and anti-p53 antibodies are detectable in serum of certain cancer patients. Increased levels of some of these products were reported in lung cancer patients after occupational asbestos exposure, after exposure to polycyclic aromatic hydrocarbons or vinyl chloride. This molecular epidemiological study investigated the use of serum EGF-R, p53-protein, and anti-p53 antibodies as biomarkers for detection of effects of radon and its decay products. METHODS: Serum EGF-R, p53-protein, and anti-53 antibodies were measured using ELISA in former uranium miners of SDAG Wismut without lung disease (n=106) and miners with Schneeberg lung cancer (n=22). They were compared with healthy subjects (n=23), patients with lung cancer not due to ionizing radiation (n=88), and patients with non-malignant lung or pleural diseases (n=50). RESULTS: No significantly elevated or decreased serum values for p53 protein, EGF-R, or anti-p53 antibodies could be found. There was no correlation of these with Working Level Months (WLM). CONCLUSIONS: p53 protein, EGF-R, or anti-p53 antibodies in serum are not useful as biomarkers for detection of lung cancer related to ionizing radiation (i.e., Schneeberg lung cancer).

p53 protein, EGF receptor, and anti-p53 antibodies in serum from patients with occupationally derived lung cancer.

The oncogene product epidermal growth factor receptor (EGF-R), the tumour suppressor gene product p53 and anti-p53 antibodies are detectable in the serum of certain cancer patients. Increased levels of some of these products were reported in lung cancer patients after occupational asbestos exposure and after exposure to polycyclic aromatic hydrocarbons or vinylchloride. In the first step, this study investigated the possible diagnostic value of serum EGF-R, p53-protein and anti-p53 antibodies, measured by an enzyme-linked immunosorbent assay, in lung tumour patients. In addition to being investigated on a molecular epidemiological basis, these parameters were examined as biomarkers of carcinogenesis, especially with regard to asbestos incorporation effects or of radon-induced lung cancers. Also, a possible effect of cigarette smoking and age dependence were studied. A total of 116 male patients with lung or pleural tumours were examined. The histological classification was four small-cell cancers, six large-cell cancers, 32 adenocarcinomas, 47 squamous carcinomas, 12 mixed lung carcinomas, five diffuse malignant mesotheliomas and ten lung metastasis of extrapulmonary tumours. Twenty-two lung cancers and all mesotheliomas were related to asbestos, 22 lung cancers were related to ionizing radiation and 61 patients had cigarette smoke-related lung cancer. Besides these patients 50 male patients with non-malignant lung or pleural diseases were included; of the latter eight subjects suffered from asbestosis. Controls were 129 male subjects without any lung disease. No significantly elevated or decreased serum values for p53 protein, EGF-R, or anti-p53 antibodies as a function of histological tumour type, age, or degree and type of exposure (asbestos, smoking, ionizing radiation) could be found. The utility of p53-protein, EGF-R and anti-p53 antibodies as routine biomarkers for screening occupationally derived lung cancers is limited.

Secreted ADP plays a central role in thrombin-induced phospholipase D activation in human platelets.

Thrombin and other agonists that induce secretion and aggregation in human platelets also activate phospholipase D (PLD), but the signaling cascade leading to activation of PLD in human platelets is not yet clear. We have determined that apyrase, which scavenges ADP secreted during platelet activation, is able to block or reduce the PLD activation stimulated by low (0.1 U/ml or less) or high (0.3- 1.0 U/ml) concentrations of thrombin, respectively. Neither ADP (up to 100 microM) nor its more potent analogue 2-methylthio-ADP (up to 100 microM), however, are able to stimulate PLD alone, and even the addition of fibrinogen, which results in platelet aggregation, is not sufficient for PLD activation. In contrast, ADP is able to stimulate PLD in the presence of low concentrations of thrombin that alone have little or no effect, suggesting ADP may play an amplifying role in platelet PLD activation. This hypothesis is supported by the finding that the purinergic receptor antagonist ARL 66096, an ATP analogue, reduces in a concentration-dependent fashion the PLD response to thrombin (IC50=28 nM with 0.1 U/ml thrombin). ARL 66096 also abolishes the PLD activation by ADP observed in the presence of low concentrations of thrombin, confirming that the antagonist inhibits an ADP-dependent component of the response. In addition, the thromboxane A2 receptor agonist U46619 activates PLD, and this response is markedly reduced by ARL 66096. Concomitantly, phosphorylation of the protein kinase C substrate pleckstrin in response to thrombin or U46619 is partially or totally inhibited by ARL 66096, respectively, consistent with ADP stimulation of protein kinase C being involved in the PLD response to these agonists. Based on these findings, we conclude that ADP secretion and activation of purinergic ADP receptors is an important amplification mechanism in the signal transduction pathways leading to PLD activation in human platelets.

Identification of phosphorylated proteins from thrombin-activated human platelets isolated by two-dimensional gel electrophoresis by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) and liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS).

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is a powerful tool to separate complex protein mixtures including whole cell lysates. In combination with immunoblotting techniques or radioactive labeling techniques it is a fast and convenient way to demonstrate the presence of certain proteins or protein modifications. With the development of extremely sensitive analytical techniques such as matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) or electrospray ionization (ESI)-MS, it has become possible to use 2-D gels not only as an analytical but also as a preparative tool. Starting with a number of spots excised from 2-D gels, a protein can be identified using different strategies involving enzymatic cleavage of the protein in the gel matrix, elution of the resulting peptides and analysis of these peptides by mass spectrometry. The obtained peptide mass fingerprint or fragment ion spectra from peptides can be used to screen protein or nucleic acid databases in order to identify the protein. We have used the techniques described above to identify proteins from human platelets which change their phosphorylation state following activation of platelets by thrombin. Platelets were radioactively labeled with [32P]orthophosphate and stimulated. Several protein spots in the observed range of 10-80 kDa and an isoelectric point of 3-10 showed a significant increase or decrease in phosphorylation. We present the results from the investigation of a spot group representing different isoforms and phosphorylation states of myosin light chain.

Platelet phospholipase D is activated by protein kinase C via an integrin alpha IIb beta 3-independent mechanism.

Blood platelets contain phospholipase D (PLD) that is rapidly activated following platelet stimulation. It is currently unclear, however, where PLD fits into the signalling cascade that leads to aggregation and secretion. Therefore we investigated the mechanism of activation of PLD in human platelets, using the formation of the PLD-specific product phosphatidylethanol as a measure of PLD activity. PLD was activated by a number of platelet agonists that also cause the activation of protein kinase C, including thrombin, collagen, the Ca2+ ionophore A23187 and the thromboxane A2-mimetic U46619. Phorbol 12-myristate 13-acetate (PMA), a direct activator of protein kinase C, also increased PLD activity. A selective inhibitor of protein kinase C, Ro-31-8220, totally blocked the stimulation of PLD by thrombin or PMA under conditions in which it also inhibited phosphorylation of pleckstrin, the major protein kinase C substrate in platelets. Ro-31-8220 additionally inhibited A23187-stimulated PLD activity, indicating that Ca2+ activation of PLD also occurs via a protein kinase C-dependent pathway. In the presence of the fibrinogen antagonist peptide RGDS, which inhibits fibrinogen binding to integrin alpha IIb beta 3 and allows little or no aggregation to occur, thrombin- and PMA-stimulated PLD activity was still observed, indicating that PLD activation is not simply a consequence of platelet aggregation. Furthermore, these agonists were able to stimulate PLD in platelets from a Glanzmann's thrombasthenia type I patient lacking the integrin alpha IIb beta 3 complex, which indicates that activation of PLD is also independent of the recruitment of integrin alpha IIb beta 3. Taken together, our results show that PLD is activated by a pathway involving protein kinase C, and suggest that PLD might be involved in signal transduction events occurring upstream of integrin alpha IIb beta 3 activation and fibrinogen binding, which are prerequisites for full platelet aggregation.

Rapid protein tyrosine phosphorylation in the cytoskeleton of stimulated human platelets.

Upon activation platelets show elevated protein tyrosine phosphorylation, and translocation of the protein tyrosine kinase pp60c-src from the plasma membrane to the cytoskeleton occurs. We therefore investigated whether tyrosine phosphorylation also increases in the cytoskeletal compartment. Here we show that almost identical patterns of phosphotyrosine-containing proteins are detectable in the cytoskeleton after platelet stimulation with compounds that directly (phorbol 12-myristate, 13-acetate) or indirectly (thrombin, vasopressin, collagen, ADP) activate protein kinase C. The apparent molecular masses of the proteins phosphorylated at tyrosine residues are 145, 130, 100, 85, 80, 60, 56, 54 and 38 kDa. Elevation of cyclic AMP by prostaglandin E1 had no effect. Concentrations of thrombin as low as 0.01 units per ml are able to cause tyrosine phosphorylation of multiple proteins. The time course of protein tyrosine phosphorylation for thrombin- and vasopressin-stimulated platelets revealed a rapid increase in the cytoskeleton within 5 to 20 s following activation consistent with a role in early events of platelet function.

Inhibition of phospholipase D of human platelets by protein tyrosine kinase inhibitors.

Protein tyrosine kinases (PTKs) of the src family are thought to play an important role in platelet signal transduction, but little is known about the targets of these enzymes in platelets. We determined that exposure of human platelets to pervanadate, an inhibitor of protein tyrosine phosphatases, caused an increase in the activity of phospholipase D (PLD), an enzyme that might be involved in signaling events leading to aggregation or secretion. To further investigate whether tyrosine phosphorylation is a step in the pathway of activation of PLD in response to thrombin, we tested the effects of a series of PTK inhibitors on the activity of platelet PLD. PLD was activated in response to 0.3 U/ml thrombin, and this activation was reduced by several of the PTK inhibitors, especially genistein, methyl 2,5-dihydroxycinnamate (MDHC), ST271, and the tyrphostins A25 and A47. In saponin-permeabilized platelets, we observed a marked inhibition of GTP-gamma S-stimulated PLD by many of the PTK inhibitors, consistent with the possibility that PTKs are involved in the regulation of PLD activity by a G-protein or small GTP-binding protein. MDHC did not affect PLD activity in permeabilized cells, which suggests that this compound might inhibit PLD in intact platelets via another pathway. The inhibitors were also tested for their effects on the phosphorylation of a peptide substrate of src-family PTKs in a platelet membrane preparation and in permeabilized platelets. Several of the compounds partially inhibited peptide phosphorylation in the membrane preparation and in permeabilized platelets, most notably ST271, ST638, and tyrphostin A25.(ABSTRACT TRUNCATED AT 250 WORDS)

The protein tyrosine kinase pp60c-src is activated upon platelet stimulation.

Stimulation of human platelets causes a dramatic increase in phosphorylation of various proteins at tyrosine residues. The abundance of protein tyrosine kinases of the src-family in platelets, particularly pp60c-src, suggests an important role of these kinases in response to stimulation events. We have shown that pp60c-src is activated on agonist-induced platelet stimulation with respect to its substrate affinity. This was accompanied by phosphorylation of pp60c-src at Ser-12, a residue which is phosphorylated by PKC. Inhibition of PKC with a specific inhibitor, Ro-31-8220, suppressed thrombin-induced translocation of pp60c-src to the cytoskeleton. On the basis of our data, we suggest that the cytoskeletal association of pp60c-src is dependent on phosphorylation of pp60c-src at Ser-12 by PKC. Phosphorylation at Ser-12 in the membrane-binding domain might be the signal that displaces pp60c-src from the plasma membrane and, accompanied with the increased substrate affinity, facilitates phosphorylation of putative substrates.

Heparin-associated thrombocytopenia: the effects of various intravenous IgG preparations on antibody mediated platelet activation--a possible new indication for high dose i.v. IgG.

The immunologic type of heparin-associated thrombocytopenia (HAT) is caused by antibodies which activate platelets via the Fc-receptor in the presence of polysulfated oligosaccharides. The antigen is formed by a releasable platelet protein (in many cases PF4) complexed to heparin. Since the role of GP IIb/IIIa in platelet activation by HAT antibodies is controversial, we investigated platelet activation by antibodies related to HAT. We used normal platelets and platelets from a patient with Glanzmann's thrombasthenia (GT) lacking GP IIb/IIIa. Heparin and sera from patients with HAT stimulated GT platelets in the same manner as determined by 14C-serotonin release and the changes in phosphorylation of p20 and p47. Platelet activation could be inhibited by an anti FcRII monoclonal antibody (IV. 3, Fab-fragments), and by Fc-fragments, but not by F(ab')2-fragments of human IgG. The effect of four different, commercially available preparations of intact i.v. IgG on the platelet activation by six HAT sera was investigated by 14C-serotonin release. The inhibitory effect was strongly dependent upon the manufacturing process. At a concentration of 20 mg/ml only IgG that had been subjected to low pH and traces of pepsin sufficiently inhibited platelet activation. IgG treated with polyethylenglycol or sulfitolysis was less effective, whereas beta-propiolactone-treated IgG almost completely lost the ability to inhibit platelet activation by antibodies related to HAT. We conclude that inhibition of GP IIb/IIIa-fibrinogen interaction is insufficient for preventing platelet activation in HAT.(ABSTRACT TRUNCATED AT 250 WORDS)

[Fc receptor-dependent platelet activation results independently of glycoprotein complex IIb/IIIa]. / Die Fc-Rezeptor-abhängige Thrombozytenaktivierung erfolgt unabhängig vom Glykoproteinkomplex IIb/IIIa.

Platelets of a patient with Glanzmann's thrombasthenia revealed the same activation pattern when stimulated with antibodies of patients with heparin-associated thrombocytopenia (HAT) or immune complexes. This was investigated by the 14C-serotonin release test and by changes in phosphorylation of p20 and p47. Platelet activation by HAT antibodies was completely inhibited by a moab against the platelet FcRII (IV. 3) and by Fc fragments of human IgG but not by F(ab)2 fragments. We conclude that platelet activation via the FcRII occurs independently of the glycoprotein complex IIb/IIIa. Therapeutical approaches targeting GP IIb/IIIa-fibrinogen interaction seem to be not appropriate in HAT.

Substrate affinity of the protein tyrosine kinase pp60c-src is increased on thrombin stimulation of human platelets.

Human blood platelets contain high levels of non-receptor protein tyrosine kinases of the Src family, particularly pp60c-src, suggesting an important role for these enzymes in platelet physiology. Indeed, in response to various agonists of platelet function, a number of proteins become phosphorylated at tyrosine residues. However, no enzymic activation of an Src-related tyrosine kinase has yet been shown in platelets. In searching for the kinase(s) responsible, we found that all agonists tested that directly or indirectly activate protein kinase C in platelets (phorbol 12-myristate, 13-acetate, thrombin, vasopressin, collagen, calcium ionophore A23187) increased the overall activity of pp60c-src determined by IgG phosphorylation in an immunocomplex assay in the presence of low ATP concentrations. On the other hand, elevation of cyclic AMP directly by forskolin or indirectly by prostaglandin E1, or elevation of cyclic GMP by sodium nitroprusside did not significantly affect the activity of the enzyme. To substantiate the differences in enzyme activity, we determined Km and Vmax, values of pp60c-src from resting and thrombin-stimulated platelets. Thrombin treatment increased substrate affinity of pp60c-src as indicated by a 2- to 3-fold decrease in the Km values for ATP and the exogenous protein substrate casein. Vmax. values were only slightly altered under the assay conditions used. To further rule out modifications of pp60c-src in phosphorylation as a probable cause of the changed substrate affinity, we analysed tryptic phosphopeptides of immunoprecipitated, 32P-labelled pp60c-src of unstimulated and stimulated platelets. The platelet agonists listed above induced an increase in pp60c-src phosphorylation at Ser-12, which is the amino acid phosphorylated by protein kinase C. Surprisingly, we found that elevation of cyclic AMP did not affect 32P labelling of pp60c-src. On the basis of our data, we suggest that phosphorylation at Ser-12 might be one of the signal-triggering events that cause the increase in substrate affinity of pp60c-src.