Studies were performed to determine if the unstimulated platelet membrane has a site for high molecular

Because plasma kallikrein activates human neutrophils, and in plasma prekallikrein (PK)

Abstract. Fibrinogen inhibited ~25I-high molecular weight kininogen (HMWK) binding and displaced bound J25I-HMWK from neutrophils. Studies were performed to determine whether fibrinogen could bind to human neutrophils and to describe the HMWK-fibrinogen interaction on cellular surfaces. At 4°C, the binding of ~2H-fibrinogen to neutrophils reached a plateau by 30 min and did not decrease. At 23 and 37°C, the amount of t25I-fibrinogen bound peaked by 4 min and then decreased over time because of proteolysis of fibrinogen by human neutrophil elastase (HNE). Zn++ (50 #M) was required for binding of ~25I-fibrinogen to neutrophils at 4°C and the addition of Ca ++ (2 mM) increased the binding twofold. Excess unlabeled fibrinogen or HMWK completely inhibited binding of ~25I-fibrinogen. Fibronectin degradation products (FNDP) partially inhibited binding, but prekallikrein and factor XII did not. The binding of ~25I-fibrinogen at 4°C was reversible with a 50-fold molar excess of fibrinogen or HMWK. Binding of ~25I-fibrinogen, at a concentration range of 5-200/~g/ml of added radioligand, was saturable with an apparent Kd of 0.17/~M and 140,000 sites/cell. The binding of ~25I-fibrinogen to neutrophils was not inhibited by the peptide RGDS derived from the ot chain of fibrinogen or by the mAb 10E5 to the platelet glycoprotein IIb/IIIa heterodimer. Fibrinogen binding was inhibited by a 3,-chain peptide CYGHHLGGAKQAGDV and by mAb OKM1 but was not inhibited by OKM10, an mAb to a different domain of the adhesion glycoprotein Mac-1 (complement receptor type 3 [CR3]). HMWK binding to neutrophils was not inhibited by OKM1. These observations were consistent with a further finding that fibrinogen is a noncompetitive inhibitor of tzSI-HMWK binding to neutrophils. Fibrinogen binding to ADP-stimulated platelets was increased twofold by Zn +÷ (50 #M) and was inhibited by HMWK. These studies indicate that fibrinogen specifically binds to the C3R receptor on the neutrophil surface through the carboxy terminal of the 3,-chain and that HMWK interferes with the binding of fibrinogen to integrins on both neutrophils and activated platelets.

The unstimulated platelet surface contains a specific and saturable binding site for high molecular weight kininogen (HK) and low molecular weight kininogen (LK). Investigations were performed with purified heavy and light chains of HK to determine which portion(s) of the HK molecule binds to the platelet surface. Purified 64-Kd heavy chain of HK and 56-Kd light chain of HK, independently, inhibited 125I-HK binding to unstimulated platelets with a 50% inhibitory concentration (IC50) of 84 nmol/L (apparent Ki, 30 nmol/L) and 30 nmol/L (apparent Ki, 11 nM), respectively. The ability of each of the purified chains of HK to independently inhibit 125I-HK binding was not due to cleavage, reduction, and alkylation of the protein, because two-chain HK, produced by treating HK the same way as purifying the separate chains, inhibited binding similarly to intact HK. Further, purified LK alone inhibited 125I-HK binding to platelets (Ki, 17 +/- 1 nmol/L, n = 7). The 64-Kd heavy chain of HK was a competitive inhibitor on a reciprocal plot of 125I-HK-platelet binding with an apparent Ki of 28 +/- 6 nmol/L (n = 4). Independently, purified 56-Kd light chain of HK was also found to be a competitive inhibitor of 125I-HK-platelet binding, with an apparent Ki of 11 +/- 3 nmol/L (mean +/- SEM, n = 4). These indirect studies indicated that HK binds to platelets by two portions of the molecule, one on the heavy chain and another on the light chain. Studies with 125I-light chain of HK showed that it specifically bound directly to platelets in the presence of zinc, since it was blocked by HK, light chain of HK, or EDTA, but not by LK, C1s, C1 inhibitor, plasmin, factor XIII, or fibrinogen. Purified light chain of HK did not inhibit direct 125I-LK binding to platelets. HK was found to bind to platelets in an unmodified form. HK bound to platelets was cleaved by plasma or urinary kallikrein at a slower rate than the same concentration of soluble HK or HK bound and subsequently eluted from the platelet surface. Cleavage of platelet-bound HK correlated with bradykinin liberation. These studies indicate that HK has two domains on its molecule that bind to platelets. Further, platelet-bound HK is protected from kallikreins' proteolysis. This latter finding suggests that cell binding may modify the rate of bradykinin liberation from HK.

Sleep disordered breathing (SDB) and obstructive sleep apnea (OSA) during pregnancy are growing health concerns because these conditions are associated with adverse outcomes for newborn infants. SDB/OSA during pregnancy exposes the mother and the fetus to intermittent hypoxia. Direct exposure of adults and neonates to IH causes neuroinflammation and neuronal apoptosis, and exposure to IH during gestation (GIH) causes long-term deficits in offspring respiratory function. However, the role of neuroinflammation in CNS respiratory control centers of GIH offspring has not been investigated. Thus, the goal of this hybrid review/research article is to comprehensively review the available literature both in humans and experimental rodent models of SDB in order to highlight key gaps in knowledge. To begin to address some of these gaps, we also include data demonstrating the consequences of GIH on respiratory rhythm generation and neuroinflammation in CNS respiratory control regions. Pregnant rats were exposed to daily intermittent hypoxia during gestation (G10-G21). Neuroinflammation in brainstem and cervical spinal cord was evaluated in P0-P3 pups that were injected with saline or lipopolysaccharide (LPS; 0.1mg/kg, 3h). In CNS respiratory control centers, we found that GIH attenuated the normal CNS immune response to LPS challenge in a gene-, sex-, and CNS region-specific manner. GIH also altered normal respiratory motor responses to LPS in newborn offspring brainstem-spinal cord preparations. These data underscore the need for further study of the long-term consequences of maternal SDB on the relationship between inflammation and the respiratory control system, in both neonatal and adult offspring.