Partial isolation and function of the prostacyclin regulating plasma factor

Rat aortic rings stop producing prostacyclin upon prolonged washing in buffer. This 'exhaustion' is caused by inhibition of cyclo-oxygenase, since these rings still convert cyclic endoperoxides but not arachidonic acid into prostacyclin, and most probably is due to high concentrations of peroxides: it can be accelerated by H2O2 or by interrupting the glutathione cycle, while it is delayed by reduced glutathione. Incubation of exhausted rings in human plasma or in a plasma filtrate restores to some extent prostacyclin formation. This filtrate, in particular from uraemic subjects, also inhibits the H2O2 initiated oxidation of guaiacol by ram seminal vesicle microsomes or horseradish peroxidase. The prostacyclin regulating plasma factor has been partially purified and identified as a stable and very polar molecule of mol. wt. 300-400, able to reactivate prostacyclin generation by exhausted rings. We suggest that one or more low mol. wt. plasma components prolong vascular prostacyclin formation by acting as reducing cofactor for cyclo-oxygenase peroxidase. The main physiological role of this plasma activity is probably to protect the vascular prostacyclin forming system from exhaustion during persistent irritation.     

血栓性血小板减少性紫癜患者vWF裂解蛋白酶抗原和活性的检测及意义

目的研究血管性血友病因子裂解蛋白酶(ADAMTS13)抗原含量和活性在血栓性血小板减少性紫癜(TTP)患者及遗传性 TTP 家族突变携带者中变化的情况。方法用残余胶原结合实验(RCBA)检测13例 TTP 患者共28份血浆标本[含血浆置换(PE)前后]及10例携带者的 ADAMTS13活性;用新近建立的三抗体夹心酶联免疫反应法检测标本的 ADAMTS13抗原含量。结果正常对照组 ADAMTS13含量为(600.93±145.36)mU/ml(设白种人混合血浆的 ADAMTS13抗原含量为1000mU/ml),活性为(74.79±11.81)%。遗传性 TTP 患者 ADAMTS13抗原含量和活性治疗前和发病间期均明显减低,PE 后恢复;其家族中携带者 ADAMTS13抗原含量为(331.40±109.85)mU/ml,活性为(66.79±12.82)%(与对照组比较,P 值分别<0.01和>0.05);原发性 TTP 患者 PE 前 ADAMTS13抗原含量为(98.7±82.08)mU/ml,活性为(22.23±19.07)%(与对照组比较,P 值均<0.01);PE 后ADAMTS13 抗原含量为(449.4±232.33)mU/ml,活性为(60.92±22.33)%(与对照组比较,P 值分别<0.01和>0.05);1例继发性 TTP 患者 PE 后 ADAMTS13抗原含量远高于正常,活性仅为6.00%结论治疗前的 TTP 患者 ADAMTS13抗原含量和活性均明显减低。大多数患者两指标变化趋势一致,也有个别患者两指标变化趋势相反,前者可能因为遗传因素或体内免疫系统的廓清作用,后者可能因为抗 ADAMTS13抗体仅抑制了 ADAMTS13的活性而未影响其抗原的含量或其他未知原因所致。     

Anti-vWf Antibodies Induce GPIbα and FcγRII Mediated Platelet Aggregation Only at Low Shear Forces

Background/Methods: Anti-von Willebrand factor (vWf) antibody mediated platelet activation was studied using 2 monoclonal anti-vWf antibodies promoting the binding of vWf to GPIb: 1C1E7 (IgG2a) reacting with the vWf N-terminus and 75H4B12 (IgM), characterized in this paper and studied in association with 1C1E7. Results: 75H4B12 binds to an N-terminal epitope in vWf, different from that reacting with 1C1E7. When com-bined, 1C1E7 and 75H4B12 promoted vWf binding to isolated GPIb under static conditions, even in the absence of ristocetin or botrocetin, and induced platelet aggregation synergistically in the presence of zero to subthreshold ristocetin concentrations. Specific inhibitors of GPIb-vWf interactions prevented vWf binding to GPIb in ELISA and during platelet aggregation. In addition, the 1C1E7 dependent platelet aggregation involved Fc receptor mediated platelet activation, a phenomenon even more pronounced when 1C1E7 and 75H4B12 were combined. A 75H4B12 binding phage expressing a peptide homologous with vWf sequence 88–95 neutralized the antibody induced platelet activation. However, at arterial shear rates, both 1C1E7 and 75H4B12 potently prolonged cartridge closure times in the PFA-100, compatible with inhibition of platelets by vWf, unfolded by the combined action of shear stress and antibodies. Conclusions: We conclude that antibodies directed against different epitopes in the N-terminus of vWf modify the folded vWf structure synergistically and enhance A1 domain mediated vWf binding to platelet GPIb at low shear forces. In addition, once platelet-bound, IgG antibodies potently activate platelets via the FcII receptor. Thus, such antibodies may promote immune mediated thrombosis at low shear rates, typical of the venous circulation. In contrast, at arterial shear rates, anti-vWf antibodies may rather compromise platelet function following enhanced binding of the unfolded vWf multimers to platelets, shielding platelets from interacting with subendothelial and soluble ligands.     

Prostacyclin production by whole blood from children: impairment in the hemolytic uremic syndrome and excessive formation in chronic renal failure

The capacity of leukocytes to produce prostacyclin (PGI2) from endogenous and from platelet-derived endoperoxides was tested in whole blood. During the acute phase of the hemolytic uremic syndrome (H.U.S.), the PGI2-production was lower than the controls, whereas the blood from children with chronic renal failure produced higher amounts. Production of PGI2 by blood from children 3/12 to 6 years after the acute phase of H.U.S. was normal, as was the case with blood from their parents. Furthermore, in two H.U.S.-patients studied serially, the decreased PGI2-production capacity normalized 2 1/2 months after the acute phase.     

A thromboxane synthetase inhibitor reorients endoperoxide metabolism in whole blood towards prostacyclin and prostaglandin E2

During incubation of citrated blood at 37°C the levels of 6-ketoprostaglandin F₁ (6-keto PGF₁) and prostaglandin E₂ (PGE₂) remain constant, but rise markedly within one minute after the addition of collagen, particularly when thromboxane synthetase is blocked. The amount of 6-keto PGF₁ formed is dose-dependent for both collagen and the thromboxane synthetase inhibitor (UK-37,248). Moreover, the number of platelets will determine the extent of the 6-keto PGF₁ jump, that does not occur when blood is drawn after aspirin ingestion. The production of 6-keto PGF₁ in function of time is composed of a fast platelet-related (intercept) and a slower probably leukocyte-dependent contribution (slope). In the absence of UK-37,248 the intercept is 115 ± 85 pg/ml, the slope is 12.9 ± 7.7 pg/min/ml whereas in the presence of the thromboxane synthetase inhibitor they are 411 ± 177 pg/ml and 56.2 ± 25 pg/min/ml respectively. The present findings indicate that a thromboxane synthetase inhibitor, by not only reducing thromboxane A₂ production but also enhancing prostacyclin generation when blood is exposed to thrombogenic stimuli such as collagen, should be superior to aspirin as an antithrombotic agent, although possible interference by enhanced PGE₂ production should be taken into account.     

Fc-independent cross-linking of a novel platelet membrane protein by a monoclonal antibody causes platelet activation

A monoclonal antiplatelet antibody (MA-13G8E1) is described that dose- dependently induces platelet aggregation and serotonin release in an Fc- independent fashion. Whereas platelets were equally aggregated by F(ab')2 fragments of this monoclonal antibody (MoAb), its Fab fragments, on the other hand, were inactive, indicating that divalent interaction is an essential requirement to induce platelet activation by MA-13G8E1. In addition, we could show that platelet epitope cross- linking by MA-13G8E1 occurred on the same platelet. MA-13G8E1 stimulated platelet phospholipase C (PLC) and induced activation of protein kinase C (PKC), both of which were almost unaffected by aspirin pretreatment. Furthermore, PLC activation appeared to be a direct antibody-mediated effect, since intracellular Ca2+ rises were not inhibited by EGTA, cytochalasin B, or aggregation-blocking MA-16N7C2 (antiglycoprotein [anti-GP]IIb/IIa). The MA-13G8E1 antigen is constitutively expressed on resting platelets of different species (7,100 +/- 800 molecules per human platelet), but not on other cell types tested. Both immunoprecipitation and affinity isolation by MA- 13G8E1 showed two low-molecular weight proteins (45 and 36 kD), having slightly acidic isoelectric pH levels (4.5 to 5.5) and forming multimolecular complexes. In conclusion, we found an MoAb that is able to induce platelet activation in an Fc-independent fashion. The mechanism involves cross-linking of a hitherto undescribed platelet membrane protein, leading to PLC and PKC stimulation.     

Manipulation of the local thromboxane and prostacyclin balance in vivo by the antithrombotic compounds dazoxiben, acetylsalicylic acid and nafazatrom

We have developed an experimental model for the study of local prostaglandin production by platelets and the vessel wall following stimulation ‘in vivo’. A nylon thread was inserted into the external jugular vein of rabbits; its presence did not induce an occluding thrombus. Thromboxane (TXB₂) values in the blood, sampled through the facial vein, immediately distal to the stimulus, rose and remained high for at least 4hr, while 6-keto prostaglandin (PG) F_1α levels, after a first increase, gradually returned to normal (‘exhaustion’ of the endothelial cells?). No changes were observed in the contralateral jugular vein without thread.After infusion via the femoral vein of 10 mg/kg dazoxiben, a thromboxane synthetase inhibitor, local TXB₂ production was completely abolished, whereas 6-keto PGF_1α formation no longer returned to basal values, but tended to increase. This leads to the conclusion that upon inhibition of TXB₂ formation endoperoxide metabolism is reoriented ‘in vivo’ towards prostacyclin, and this mainly at the site where platelets are activated.Injection of 100 mg/kg lysine acetylsalicylic acid resulted in complete inhibition of TXB₂ and 6-keto PGF_1α formation, the latter, however, slowly recovering with time.The administration of nafazatrom to the animals did not influence the local TXB₂ changes, but partially prevented the decline of 6-keto PGF_1α with time. The antithrombotic properties of this drug thus could be related to protection of the endothelial cells from ‘exhaustion’.