Carbon monoxide releasing molecule-2 inhibition of snake venom thrombin-like activity: novel biochemical "brake"?

Abstract

A complication of defibrinogenation therapy with snake venom enzymes such as ancrod is hypofibrinogenemia associated bleeding secondary to no human-derived inhibitor being available to inactivate or diminish the activity of such enzymes. Of interest, ancrod contains a critical histidine residue without which enzymatic activity is inhibited, and carbon monoxide has been demonstrated to inhibit biomolecular function by interacting with histidine moieties in ion channels.

We tested the hypothesis that exposure of three different snake venoms containing serine proteases with thrombin-like activity (which included ancrod) to carbon monoxide derived from carbon monoxide releasing molecule-2 would diminish their effects on plasmatic coagulation as assessed by thrombelastography. In the case of the Malayan pit viper and Eastern diamondback rattlesnake venoms, carbon monoxide diminished the effects of thrombin-like activity.

In contrast, timber rattlesnake venom demonstrated enhancement of "thrombin-generating" activity with simultaneous loss of thrombin-like activity in response to carbon monoxide exposure. These findings may serve as the rational basis for not just continuing to investigate the potential of snake venom enzymes as clinical defibrinogenating agents, but to also to assess the potential to stop such agents from becoming a catalytic "runaway train" by judicious application of a biochemical "brake" such as carbon monoxide.

PMID:

27787696

DOI:

10.1007/s11239-016-1442-4

Thrombin Activity Associated with Neuronal Damage during Acute Focal Ischemia

Mechanisms of ischemic neuronal and vascular injury remain obscure. Here we test the hypothesis that thrombin, a blood-borne coagulation factor, contributes to neurovascular injury during acute focal ischemia. Stroke was induced in adult Sprague Dawley rats by occluding the middle cerebral artery. Intra-arterial thrombin infusion during ischemia significantly increased vascular disruption and cellular injury. Intravenous infusion of argatroban, a direct thrombin inhibitor, alleviated neurovascular injury. Immunostaining showed thrombin on neurons in the ischemic core.

Using an activatable cell-penetrating peptide engineered to detect thrombin activity, we discovered that thrombin proteolytic activity was specifically associated with neuronal damage during ischemia. Protease activated receptor-1, the presumptive thrombin receptor, appeared to mediate ischemic neurovascular injury. Furthermore, rats receiving thrombin during ischemia showed cognitive deficit, whereas rats receiving argatroban retained intact learning and memory. These results suggest a potential role for thrombin contributing to neurovascular injury and several potential avenues for neuroprotection.


The Journal of Neuroscience, 30 May 2012, 32(22): 7622-7631; doi: 10.1523/​JNEUROSCI.0369-12.2012 

PubMed citation
Articles by Chen, B.
Articles by Lyden, P. D.

DNA–enzyme conjugate with a weak inhibitor that can specifically detect thrombin in a homogeneous medium

Abstract
We present the DNA-assisted control of enzymatic activity for the detection of a target protein using a new type of DNA–enzyme conjugate. The conjugate is composed of an enzyme inhibitor to regulate enzyme activity and a DNA aptamer to be responsive toward the analyte protein. Glutathione S-transferase (GST) and thrombin were selected as a model enzyme and an analyte protein. A hexahistidine tag was genetically attached to the C terminus of the GST, and the 5′ end of an oligonucleotide was conjugated with nitrilotriacetic acid (NTA) for the site-specific conjugation of the DNA with the GST based on a Ni2+ complex interaction. We found that fluorescein acted as a weak inhibitor of GST and succeeded in the regulation of GST activity by increasing the local concentration of the weak inhibitor by the hybridization of a 3′-end fluorescein-modified DNA. The catalytic activity of the DNA aptamer–enzyme conjugate showed a dose-dependent response to thrombin, indicating that the GST activity was clearly recovered by the binding of the DNA aptamer to thrombin. The current system enables the sensitive and specific detection of thrombin simply by measuring the enzymatic activity in a homogeneous medium.

Analytical Biochemistry
Volume 414, Issue 1, 1 July 2011, Pages 103-108