Vascular reactivity in sepsis and platelet dysfunction in septic shock.

Sepsis remains an important global cause of morbidity and mortality. Sepsis can be complicated by pathological vasodilation causing cardiovascular septic shock. The present study identifies that dysfunction of the RhoA/Rho-kinase (ROK) signalling pathway in vascular smooth muscle cells contributes to vasomotor dysfunction in sepsis. ROK inhibits myosin light chain phosphatase (MLCP) through Thr855 phosphorylation of MYPT, the L30 kDa myosin binding regulatory subunit of MLCP. MLCP dephosphorylates myosin light chain (LC₂₀) inhibiting the acto-myosin cross-bridge cycling underpinning vasoconstriction or platelet contraction. ROK dependent MLCP inhibition therefore favours vasoconstriction and can be indexed by Thr855-MYPT phosphorylation. Western blot analysis identified that Thr855 phosphorylation of MYPT was reduced in arterial segments isolated from a murine caecal ligation and puncture model of sepsis. Wire myography yielded data consistent with reduced contractile responses to thromboxane A₂ receptor stimulation, high [K⁺] mediated depolarisation and direct PKC stimulation. α₁₋ adrenergic receptor mediated vasoconstriction was similar in septic and non-septic animals, possibly reflecting the multiple mechanisms by which α₁₋ adrenergic agonists elicit vasoconstriction. Certain bacterial toxins and inflammatory mediators have the potential to attenuate ROK signaling; our data suggest therapeutic benefit of agents that promote MLCP inhibition or which vasoconstrict independent of the RhoA/ROK pathway. Current vasopressor strategies for septic shock primarily rely upon catecholamine therapy. However, there is interest in administration of vasopressin, an endogenous vasopressor inappropriately suppressed in septic shock. It is proposed that vasopressin mediates Ca²⁺ sensitisation through ROK mediated inhibition of MLCP, however, neither vasopressin dependent Ca²⁺ sensitisation nor Thr855 MYPT phosphorylation have been directly identified. In permeabilised rat caudal artery Ca²⁺ sensitisation was observed and found to depend at least partly upon PKC signaling. In contrast, stimulation with arginine vasopressin (AVP) was not associated with Thr855 MYPT phosphorylation despite the ROK inhibitor Y27632 attenuating vasopressin dependent vasoconstriction. These data support clinical evaluation of vasopressin therapy targeted to cases of septic shock arising from organisms capable of producing toxins, which neutralise RhoA/ROK. Furthermore, the data suggest either an MLCP independent vasoconstrictor role for ROK or ROK independent action of Y27632. Sepsis is also complicated by coagulopathy promoting both thrombosis and haemorrhage. Data regarding platelet function in sepsis is equivocal and absent in the specific subset of patients with septic shock. Recognising the importance of platelet contraction in thrombus formation and suggested similarities between vascular smooth muscle and platelet contraction we aimed to identify whether platelet contractile dysfunction contributed to impaired platelet aggregation in septic shock. Whole blood impedance aggregation was impaired in patients suffering from septic shock; deficits in aggregation correlated with illness severity. Impaired platelet aggregation was not associated with biochemical evidence of contractile dysfunction: neither Ser19-LC₂₀ nor Thr855-MYPT phosphorylation differed between septic shock and non-septic patients. These data indicate that therapeutic strategies to restore platelet function in septic shock might more profitably focus on platelet adhesion and secretion. These studies identify MLCP inhibition as a potential therapeutic avenue to ameliorate vascular smooth muscle, but not platelet, function in septic shock. Vasopressin might provide particularly effective vasoconstriction when targeted to cases of septic shock associated with disrupted ROK/MLCP integrity.Thesis (Ph.D.) -- University of Adelaide, School of Medical Sciences, 201