Neutralization of the antiheparin activity of platelet factor 4 by a monoclonal antibody.

We have produced a panel of monoclonal antibodies (mAbs) against rabbit platelet factor 4 (PF4). Two of these mAbs have been characterized in this study. In particular the antibody called 10B2, which also recognizes the human molecule, is able to block PF4's ability to neutralize heparin in a modified Heparin-Factor Xa chromogenic assay. The inhibition appears to be more than 95% at 1:1 mAb/PF4 molar ratio both for purified rabbit and human PF4. Similar results were obtained using supernatants from stimulated human platelets (90% of inhibition at 1:1 mAb/PF4 molar ratio) or using Fab fragments from 10B2. Studies to determine the antigenic determinant against which 10B2 is directed, show that this is an assembled epitope which involves disulfide bonds of the PF4.

Effect of cloricromene during ischemia and reperfusion of rabbit hindlimb: evidence for an involvement of leukocytes in reperfusion-mediated tissue and vascular injury.

The involvement of polymorphonuclear leukocytes (PMN) in reperfusion-mediated vascular injury was studied in a model of ischemia and reperfusion in rabbit hindlimb. Ischemia was induced by 4-h occlusion of the left iliac artery followed by 4-h reperfusion. Plasma creatine kinase (CK) and lactate dehydrogenase (LDH) activities, hindlimb vascular resistance (HVR), and myeloperoxidase (MPO) activity in the postischemic extensor digitorum longus (EDL) muscle were measured to evaluate the extent of vascular and skeletal muscle injury. In addition, the ischemia/reperfusion-induced injury of the hindlimb vasculature was evaluated by electron microscopy. Ischemia and reperfusion (n = 10) was associated with an increase in CK (6,380 +/- 1,346 U/L, p < 0.05) and LDH (552 +/- 76 U/L, p < 0.05) activities which were significantly greater than those observed in sham-operated control animals (CK 1,651 +/- 207 U/L, LDH 246 +/- 14 U/L; n = 6). HVR in sham-operated animals decreased by 20 +/- 3%, but increased in the ischaemic group by 56 +/- 16% (p < 0.05). MPO activity of EDL muscle increased from 7.3 +/- 3.9 U per muscle (sham) to 28.0 +/- 5.9 U per muscle (p < 0.05) after ischemia and reperfusion. Morphologic analysis did not show any alteration in the microvascular bed of the hindlimb. Moreover, 1 mg/kg/h intravenous (i.v.) cloricromene, an antithrombotic drug that inhibits superoxide anion production as well as PMN adhesion to endothelium, reduced the increase in plasma CK and LDH and the increase in MPO and HVR observed in animals subjected to hindlimb ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional depression of isolated perfused rat heart mediated by activated leukocytes: protective effect of cloricromene.

Langendorff rat heart preparations were perfused with suspensions of human leukocytes containing approximately 65% polymorphonuclear cells (PMN). The cells were either unstimulated or activated with 1.6 x 10(-8) phorbol 12-myristate 13-acetate (PMA). Left ventricular developed pressure (LVDP), coronary flow (CF), and heart rate (HR) were recorded during PMN infusion (10 min) and for the recovery period (30 min). PMN were also pretreated with cloricromene (CLO 10-50 microM), a drug that inhibits platelet aggregation and PMN adhesion to endothelial cells (EC). Infusion of unstimulated cells did not affect cardiac function. Infusion of activated cells caused CF reduction (-44 +/- 4% at end of infusion; -24 +/- 4% at end of recovery, expressed as percentage of variation vs. basal value), LVDP decrease (-44 +/- 5% at end of infusion, -26 +/- 6% at end of recovery) endothelial damage, and leukocyte accumulation in heart as compared with hearts infused with unstimulated PMN and sham hearts. PMN accumulation was quantified as myeloperoxidase (MPO) activity (260 +/- 35, 39 +/- 6, 19 +/- 1 U/g, respectively). Superoxide dismutase (SOD 600 U/ml), catalase (2,200 U/ml), thiourea (10 mM) added to PMN suspension blunted CF decrease but not LVDP reduction and MPO increase. CLO (25-50 microM) pretreatment inhibited PMN accumulation, LVDP, and CF reduction by approximately 50%. These data suggest a role of leukocyte activation in the genesis of heart damage and raise the possibility of a pharmacologic intervention with drugs such as CLO that can interfere with this process.

Changes in vessel ultrastructure during ischemia and reperfusion of rabbit hindlimb: implications for therapeutic intervention.

Peripheral ischemia was induced in the rabbit by occlusion of the left iliac artery for 6 hr, followed by 24 hr of reperfusion. Biochemical and morphological investigations were performed to evaluate the extent of vascular and tissue injury. Blood samples for plasma enzyme determinations (creatine kinase (CK) and lactate dehydrogenase (LDH) activities) were obtained at times t = 0, t = 6, t = 30 hr. Plasma CK and LDH activities in ischemic animals were approximately twice as high as those in sham-operated animals at the end of reperfusion, although no difference was observed at the end of the period of ischemia. Morphological and morphometric analysis of extensor digitorum longus muscle from ischemic animals showed a reduction in the number of patent capillary vessels per muscle fiber (1.54 +/- 0.1 and 1.04 +/- 0.09, P < 0.05, in sham and ischemic groups, respectively; mean +/- SEM). In addition, the number of microvilli on endothelial surfaces were considerably increased in the ischemic group (0.14 +/- 0.02 and 0.41 +/- 0.01 microns -2, P < 0.001, in sham and ischemic groups, respectively). A great number of adhered leucocytes were found on the vessel surface with some leucocytes having migrated through the vessel wall. Microcirculatory damage was accompanied by the formation of microthrombi which sometimes occluded the entire vessel lumen. The infusion of 1 mg/kg/hr of cloricromene for 6 hr prevented ischemic injury in microvessels and also prevented swelling of muscle mitochondria. In the treated group the number of patent capillaries per muscle fiber was very similar to that found in sham-operated animals (1.49 +/- 0.08; P < 0.01 vs. ischemic control). In conclusion, several different cell types are involved in the pathophysiological changes which occur in microvessels during ischemia/reperfusion injury. Pharmacological interventions, which inhibit the interactions of blood cells with endothelium, may be of value in the treatment of peripheral ischemia/reperfusion injury.

Liberation of prostaglandin-like substances from the isolated coronary artery in presence of angiotensin and of 4-methylesculetin.

The aim of this work is to study the mechanism by which 4-Methylesculetin (4-ME) causes the relaxation or inhibits the Angiotensin II (ATN 2) induced contraction in the smooth muscle. The effect of 4-Me, alone or associated with Ascorbic Acid, on basal tone and ATN 2 induced contraction of isolated coronary strips have been studied. Experiments have been carried out in presence of Lysine Acetylsalicylate (LAS) and Indomethacin (IN), known inhibitors of prostaglandin-synthetase. Both LAS and IN decrease but not abolish, the 4-ME induced relaxation and suppressed the depressive effect of 4-ME on the ATN 2 dependent contraction. From R.I.A. tests results that 6-Keto PGF1 alpha (prostacyclines stable metabolite) concentration increased with the addition of 4-ME to the bath. 6-Keto PGF1 alpha concentration was drastically reduced after IN and LAS use. Therefore, it seems reasonable to conclude that 4-ME influence could be mediated by prostacyclines release in the smooth muscle.