Biochemistry of the Thrombin-Like Enzyme and Its Purification from Iranian Echis Carinatus Snake Venom: Its Interaction with Platelet Receptors

Abstract

Snake venoms are rich in valuable substances that have medical potential in the diagnosis and treatment of hemostatic diseases. The present paper was aimed at the purification and functional characterization basis of a thrombin-like enzyme and its role in the functioning of the coagulation cascade and platelet aggregation pathway. A thrombin-like serine protease was purified from the Iranian Echis carinatus venom (TLIECV), employing a one-step chromatographic procedure. This peptide was collected in high yield and purity by a single chromatographic step using RP-HPLC equipped with a C₁₈ column. This peptide showed a 3000 Da molecular weight in gel-electrophoresis. Evidence in the SDS-PAGE gel has confirmed high recovery of fraction in optimal terms. Subsequently, this peptide was identified via its intact molecular mass and peptide mass fingerprint (PMF) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). Multiple sequence alignments were performed by ClustalW, the Bioedit software. Molegro Data Modeller (MDM) 3.0 software was used to predict the putative tertiary structure of the peptide. The enzyme possessed fibrinogenolytic, procoagulant, and aggregation inducer properties. Moreover, the SDS-PAGE (12%) was applied to examine fibrinogenolytic function. The purified enzyme degraded the Aα chain of fibrinogen while the Bβ and γ chains were not digested. According to that, the deficient human plasma in factor X and normal human plasma were also coagulated by TLIECV, it takes part in the common and intrinsic routes of the coagulation cascade. These findings proved that TLIECV is a serine protease identical to procoagulant thrombin-like snake venom proteases; however, it specifically releases the Aα chain of bovine fibrinogen. Because of its function to make up for the deficiency of factor X and its platelet aggregation inducer property, TLIECV could be considered a molecular impact to reveal the hemostasis mechanisms.

Keywords: Blood coagulation; Platelet aggregation inducer; Platelet function; Snake venom; Thrombin-like.

Figure 1

a: RP-HPLC chromatography profile of the soluble venom of IEC. The column elution profile exhibited eleven peaks (F₁-F₁₁). Fractionation of crude venom by reverse-phase-HPLC using column C₁₈ equilibrated with trifluoroacetic acid (0.1%) (Solvent A). The eluted material was monitored at 280 nm. b: Confirmation of the pure peak by RP-HPLC on a C₁₈ column

Figure 2

Effect of TLIECV on clotting plasma. Data expressed as mean ± SD.a: PCT. b: RT. c: PT. d: APTT. e: TCT. f: FCA. *: No Clot

Figure 3

Coagulant activity of TLIECV

Figure 4

a: SDS-PAGE analysis of IEC crude venom. Lane 1, 2 and 3 represent protein markers, IEC crude venom (reduced), and IEC crude venom (non-reduced) respectively. b: Determination of the molecular mass of TLIEC by SDS–PAGE analysis (12.5%). Lane 1, molecular mass markers, Lane 2. TLIEC. c: TLIEC incubated with fibrinogen (3 mg/ml) at different times (1, 6, 12 and 24 h), the activity was evaluated using SDS–PAGE (12%), Lane 1, Molecular mass markers Lane 2, Fibrinogen control, Lane 3–6, different times (1, 6, 12 and 24 h), respectively.

Figure 5

Effects of the purified fraction on the platelet aggregation in washed human platelets suspension. a: 1. Washed platelets + Arachidonic Acid+ Normal saline 2. Washed platelets + Collagen + Normal saline 3. Washed platelets + ADP + Normal saline 4. Washed platelets + Thrombin + Normal saline). b: 1. Washed platelets + (TLIECV) + Normal saline 2. Washed platelets + (TLIECV+ AA) 2. Washed platelets + (TLIECV + Collagen) 2. Washed platelets + (TLIECV+ Aspirin). c: 1. Washed platelets + (TLIECV + Clopidogrel) 2. Washed platelets + (TLIECV+ Ticlopidine)

Figure 6

a: Mass spectrum profile of TLIECV. (b) PMF analysis for identification of TLIECV, (c): Alignment of the predicted amino acid sequences of TLIECV peptide with Viperidae venom protein families in protein databases. This alignment is based on the comparison with the sequence; this sequence has the most similarity with the sequence obtained from the Mascot analysis of the MASS results of the putative peptide using Bioinformatics analysis. Sequence alignment between purified peptide sequences and the closest BLAST match. (d): Predicted three-dimensional structure of TLIECV resulting from bioinformatics analysis